Meroxapols for cell culture

Abstract

To provide a cell culture medium that uses a cell culture medium additives suitable for foam reduction and for shear stress protection, and a process for culturing cells.SOLUTION: A cell culture medium comprises a meroxapol with a peak molecular weight Mp between 1000 and 8000 g / mol and a polyethylene oxide percentage between 55 and 95% (w / w).SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to the use of meloxapol, also known as "reverse poloxamer," as a cell culture medium additive. Meloxapol is suitable for foam reduction and shear stress protection.

[0002] Background of the Invention Poloxamers (especially poloxamer 188) are used in many industrial, cosmetic, and pharmaceutical applications. They are also used in cell culture medium processes. The addition of poloxamers (especially poloxamer 188) to cell culture media significantly improves cell viability. High cell viability is important for optimal protein production. Why poloxamers improve cell viability is not fully understood. It is believed that poloxamers reduce shear stress and thus protect cells from damage. Although poloxamers are nonionic surfactants, they can concentrate at the gas bubble / culture medium interface and thus protect cells from adhesion to gas bubbles and, in the event of bubble bursting, protect cells from damage. When bubbles burst, it may also reduce the impact. Some publications claim that poloxamers improve the rate of oxygen transfer from the gaseous to the liquid phase, while others refute these findings. There are also indications that poloxamers may "repair" small defects in the cell membrane.

[0003] Unfortunately, poloxamer 188 significantly contributes to the stabilization of foam produced in sparged bioreactors. Therefore, typically, an antifoaming agent is used in addition to poloxamer. Examples of antifoaming agents include agents preferably consisting of polydimethylsiloxane (PDMS) and optionally silica particles, also known as dimethicone or simethicone, for example.

[0004] In general, the addition of antifoaming agents does not solve all problems because there remains a risk of foam overflow if the antifoaming agent is added manually and the foam level is not constantly monitored. Silica-based antifoaming agents are deactivated by silica segregation during their action. In addition, antifoaming agents cannot be added in large quantities because they can become toxic at higher concentrations. Simethicone is silicone oil-based and cannot be filtered through PES membranes, and therefore cannot be sterilized by PES filtration, and thus antifoaming agent C cannot be added directly to cell culture medium preparations. The foaming problem is further exacerbated from the perspective of cultures with high cell density, as they require more oxygen, thus requiring more sparging, and therefore more foaming. Therefore, a non-foaming or low-foaming alternative to poloxamer 188 is desired.

[0005] Schmolka IR (Schmolka, Journal of the American Oil Chemists' Society (1977), 54(3), 110-16) and Murhammer (DW Murhammer, CF Goochee, Biotechnol. Prog. 1990, 6, 142-148) published data on the properties of other poloxamers in addition to poloxamer 188. They also discussed the properties of meloxapole. Although they found several candidates showing promising features regarding shear stress protection and foam stabilization, they were unable to identify an ideal candidate. Some were toxic to insect cells, and most candidates still required the addition of antifoamers.

[0006] As a result, there is still a need to find an alternative to poloxamer 188 that not only protects against shear stress but also reduces bubble formation and is non-toxic to cells.

[0007] Certain meloxapols have been found to meet all requirements regarding toxicity, foam formation, and shear stress protection. The requirement for ideal suitability in cell culture is a certain peak molecular weight combined with a defined percentage of the PEO (polyethylene oxide) portion. This results in shear stress protection comparable to poloxamer 188, i.e., comparable viable cell density, viability, and IgG productivity, while simultaneously eliminating the need for the addition of an antifoaming agent. With these meloxapols, little to no foam is formed during the process, thus eliminating the need for the addition of an antifoaming agent.

[0008] The present invention thus provides a peak molecular weight M between 1000 g / mol and 8000 g / mol. p It is directed to a cell culture medium containing meloxapol, preferably having a polyethylene oxide percentage (%EO) between 1000 g / mol and 5000 g / mol, and between 55% and 95%. In a preferred embodiment, peak molecular weight M P It is between 1800 g / mol and 3500 g / mol. In another preferred embodiment, the polyethylene oxide percentage is between 55% and 80% (w / w). In a preferred embodiment, the cell culture medium contains meloxapole in an amount between 0.1 and 10 g / L, calculated for a liquid medium. In a very preferred embodiment, the amount of poloxamer is between 0.5 and 5 g / L, calculated for a liquid medium.

[0009] In one embodiment, the cell culture medium is a chemically defined medium. In one embodiment, the cell culture medium is a dry powder or a dry condensed medium.

[0010] In another embodiment, the cell culture medium comprises at least one sugar component, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffering components, one or more coexisting factors, and one or more nucleic acid components. In a preferred embodiment, the cell culture medium does not contain any antifoaming agent.

[0011] The present invention is also directed to a process for cell culture, whereby cells are cultured in a liquid medium containing a pluronic having a peak molecular weight M between 1000 g / mol and 8000 g / mol P , preferably between 1000 g / mol and 8000 g / mol and a percentage of polyethylene oxide between 55% and 95%. Preferably, the process includes agitation and / or sparging. In a preferred embodiment, the peak molecular weight M P is between 1800 g / mol and 3500 g / mol. In another preferred embodiment, the percentage of polyethylene oxide is between 55% and 80% (w / w).

[0012] In a preferred embodiment, the amount of antifoaming agent in the liquid medium is reduced compared to a cell culture medium containing poloxamer 188 instead of pluronic as defined above, and very preferably the medium does not contain any antifoaming agent.

[0013] The present invention is therefore also directed to a method for reducing foam formation in agitated and / or sparged cell cultures, whereby cells are cultured in a liquid medium containing a pluronic having a peak molecular weight M between 1000 g / mol and 8000 g / mol P and a percentage of ethylene oxide between 55% and 95% (w / w), and foam formation is reduced compared to cells cultured under the same conditions in a cell culture medium containing poloxamer 188 instead of a pluronic having a peak molecular weight M between 1000 g / mol and 8000 g / mol P and a percentage of ethylene oxide between 55% and 95% (w / w). Preferably, no antifoaming agent is used in the method for suppressing foam formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] [Figure 1] Figure 1 shows the bubble generation during continuous air sparging at 37°C for the selected meroxapol. The meroxapol solution was prepared in cell culture medium and has a concentration of 1000 ppm. The reported curve is the output of one measurement selected from the replicates as most representative of the trend for each poloxamer. Details can be found in Example 1. [Figure 2] Figure 2 shows the bubble generation during continuous air sparging at 37°C for the selected meroxapol. The meroxapol solution was prepared in cell culture medium and has a concentration of 2000 ppm. The reported curve is the output of one measurement selected from the replicates as most representative of the trend for each poloxamer. Details can be found in Example 1. [Figure 3] Figure 3 shows the average maximum bubble height of each meroxapol solution in cell culture medium. The measurements were taken at 37°C. The values are the average of the replicates. Light gray bars, solution concentration 1000 ppm; dark gray bars, solution concentration 2000 ppm. Details can be found in Example 1. [Figure 4] Figure 4 shows the average plateau height referenced to each meroxapol solution in cell culture medium. The measurements were taken at 37°C. The values are the average of the replicates. Light gray bars, solution concentration 1000 ppm; dark gray bars, solution concentration 2000 ppm. Details can be found in Example 1.

[0015] [Figure 5] Figure 5 shows the viability (%) of CHOK1 cells during fed-batch in a rotating tube. Each value is the average of 4 replicates for each condition. [Figure 6] Figure 6 shows the viable cell density (VCD) of CHOK1 cells during fed-batch in a rotating tube. Each value is the average of 4 replicates for each condition. [Figure 7] Figure 7 shows the IgG productivity of CHOK1 cells. Each value is the average of 4 replicates for each condition. [Figure 8]Figure 8 shows the viability (%) of CHOK1 cells in fed-batch culture in rotating tubes to illustrate the need for the addition of poloxamer and / or meloxapole. Each value is the mean of 4 replicates for each condition. [Figure 9] Figure 9 shows the viability (%) of CHOK1 cells in feed batches in a bioreactor. Each data point is the mean of three replicates for each condition. Error bars represent the standard deviation of the mean. [Figure 10] Figure 10 shows the viable cell density (VCD) of CHOK1 cells during feed batches in a bioreactor. Each data point is the mean of three replicates for each condition. [Figure 11] Figure 11 shows the IgG productivity of CHOK1 cells. Each data point is the mean of three replicates for each condition. Error bars represent the standard deviation of the mean. For some data points, the error bars are not visible because the error is too small. [Figure 12] Figure 12 shows the changes in foam during supply batch culture in a bioreactor. Data points indicate the foam level in one bioreactor for each sample selected as a representative sample. Only cultures supported by poloxamer 188 required the addition of defoamer C to avoid foam overflow. Other meloxapoles stabilized only very small amounts of foam, posing no risk of overflow, and thus did not require defoamer C.

[0016] Details of Figures 5-12 are described in Example 2. In Figures 5, 6, 7, 10, 11, and 12, error bars represent the standard deviation of the mean. Data points that refer to Poloxamer 188 are connected by dashed lines to better distinguish them from other data. [Figure 13] Figure 13 shows the molecular weight distribution of meloxapole 8R6 determined by size exclusion chromatography (see Method 1). Mp indicates the peak molecular weight of a specific meloxapole.

[0017] definition Before describing the present invention in detail, it should be understood that the present invention is not limited to any particular composition or process step and may be modified in itself. Where used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to "poloxamer" includes multiple poloxamers and other similar ones. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be commonly understood by those skilled in the art relating to the present invention. The following terms are defined for the invention described herein:

[0018] The term “bioreactor,” as used herein, refers to any manufactured or operated device or system that supports a biologically active environment. In some examples, a bioreactor is a vessel or tank on which a cell culture process is carried out, involving microorganisms or biochemically active substances derived from such microorganisms. Such processes may be aerobic or anaerobic. Commonly used bioreactors are typically cylindrical, ranging in size from liters to cubic meters, and often made of stainless steel. In some embodiments described herein, a bioreactor may include a disposable component made of a material other than steel and is disposable. In some embodiments, it is a disposable bag on which a biologically active environment is maintained. The total capacity of the bioreactor is intended to be any capacity ranging from 100 mL to over 10,000 liters, depending on the particular process.

[0019] Agitated cell culture is a cell culture method in which the cell culture medium is continuously agitated with the cells in a bioreactor, either continuously or once or multiple times, during the cell culture process.

[0020] Agitation can be achieved, for example, by stirring, shaking, or agitation. In a stirred tank bioreactor, one or more agitators may be deployed depending on the geometry of the vessel. The type of agitator will be selected according to the main mixing task and requirements of the process.

[0021] Sparged cell culture is a cell culture method in which gas is introduced into a bioreactor. This is typically done using a sparger, also known as a bubbler or aeration device.

[0022] Typically, in sparged cell culture, a mixture of air, oxygen, carbon dioxide, and nitrogen is introduced into the bioreactor by a sparger. Typical spargers for cell culture processes include drilled-hole ring spargers, sintered microspargers, open-pipe spargers, and hybrid forms. Surface aeration also contributes to oxygen transport, however, the effect of surface aeration decreases during scale-up. The type of agitator and sparger in mammalian cell culture processes will often be selected according to the shear sensitivity of the cells (Nienow, AW, 2006. Reactor engineering in large scale animal cell culture, Cytotechnology, 50(1-3), p.9).

[0023] A cell culture medium according to the present invention is also a mixture of any of the components that typically maintain and / or support in vitro cell growth and / or support a particular physiological state by providing cells with at least one nutrient source. It may be a complex medium or a chemically specific medium. A cell culture medium may contain all the components necessary to maintain and / or support in vitro cell growth, or only some components, so that additional components can be added individually. An example of a cell culture medium according to the present invention is a complete medium containing all the components necessary to maintain and / or support in vitro cell growth, as well as a medium supplement or feed. In a preferred embodiment, the cell culture medium is a complete medium, a perfusion medium, or a feed medium. A complete medium, typically called a base medium, has a pH between 6.7 and 7.8. A feed medium preferably has a pH less than 8.5.

[0024] Typically, cell culture media according to the present invention are used to maintain and / or support cell growth in a bioreactor.

[0025] Feed or feed medium is a cell culture medium that, in cell culture, is added at a later stage to protect against nutrient depletion and sustain the productive phase, rather than being a base medium that supports initial growth and production. Feed medium can have higher concentrations of some components compared to base culture medium. For example, some components (such as nutrients containing amino acids or carbohydrates) may be present in feed medium at concentrations of approximately 5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×, 600×, 800×, or even approximately 1000× of the concentration in the base medium.

[0026] Mammalian cell culture media are mixtures of components that maintain and / or support the in vitro growth of mammalian cells. Examples of mammalian cells are human or animal cells, preferably CHO cells, Kos cells, I Vero cells, BHK cells, AK-1 cells, SP2 / 0 cells, L5.1 cells, hybridoma cells, or human cells.

[0027] A chemically defined cell culture medium is a cell culture medium that does not contain any chemically undefined substances. This means that the chemical composition of all chemicals used in the medium is publicly known. A chemically defined medium does not contain any yeast, animal, or plant tissue; they do not contain feeder cells, serum, hydrolysates, extracts, or digests, or other poorly defined components. A chemically undefined or poorly defined chemical component is one whose chemical composition and structure are not publicly known and can only be defined by presenting it in various compositions or by a tremendous amount of experimental effort comparable to the evaluation of the chemical composition and structure of protein-like substances such as insulin, albumin, or casein.

[0028] Powdered cell culture media or dry powdered media are cell culture media that typically result from a milling process or a freeze-drying process. It means that powdered cell culture media are granular, fine-particle media—not liquid media. The term “dry powder” may be used interchangeably with the term “powder,” however, as used herein, “dry powder” simply refers to the macroscopic appearance of granulated material and is not intended to mean, unless otherwise noted, that the material is completely mixed and solidified with solvent.

[0029] Dry granulated media are dry media resulting from wet or dry granulation processes, such as spray drying, wet granulation, or dry compaction, and typically have particle sizes greater than 0.5 mm, for example, 0.5–5 mm. Dry compaction is typically performed by a roll press. US 6,383,810 B2 discloses a method for producing moist granulated eukaryotic cell culture medium powder. The method comprises moistening a dry powder cell culture medium with a solvent, and then re-drying the moist medium to obtain dry granulated cell culture medium. Preferably, the dry granulated medium is a medium resulting from roller compaction of a dry powder medium. As used herein, the term “dry” refers simply to the macroscopic appearance of the granulated material and is not intended to mean that the material is completely mixed and solidified with solvent, unless otherwise noted.

[0030] For use in cell culture, i.e., to culture cells, liquid cell culture media are added to the cells. Dry powder or dry compressed cell culture media thus dissolve in a suitable amount of liquid, such as water or an aqueous buffer, to produce a liquid cell culture medium that can be brought into contact with the cells. Since the composition of the liquid cell culture medium directly affects the cells, the concentration of the cell culture medium is often provided in weight per liter, such as mg per liter, to define the concentration of the components in the liquid medium to be added to the cells. The amount of components in the dry powder or dry condensed medium needs to be adjusted so that the desired concentration of the components in the resulting liquid medium is achieved when dissolved in a certain amount of liquid.

[0031] According to the present invention, the cells cultured in the culture medium may be prokaryotic cells such as bacterial cells, or eukaryotic cells such as plant or animal cells. The cells may be normal cells, immortalized cells, diseased cells, transformed cells, mutant cells, somatic cells, embryonic cells, stem cells, progenitor cells, or fetal cells, any of which may be established or transformed cell lines or obtained from natural sources.

[0032] The average molecular weight determined by SEC is determined as follows: Weight-average molecular weight: Mw = Σ i N i M i 2 / (Σ i N i M i ) Number-average molecular weight: Mn n = Σ i N i M i / (Σ i N i ) Peak molecular weight: Mp p = Molecular weight at the maximum N i where N i = The number of polymer species in fraction i M i = The molecular weight of the polymer species in fraction i SEC conditions: Calibration standard: PEG (see Example (Method 1) for details) Eluent: THF Flow rate: 1 ml / min Injection volume: 100 μl Column: Particle size = 5 μm, material = styrene-divinylbenzene Temperature: 40 °C

[0033] The cell culture medium can be in the form of an aqueous liquid or, for use, in the form of a dry powder dissolved in water or an aqueous buffer. One skilled in the art can select a cell culture medium suitable for a specific, intended purpose.

[0034] The cell culture medium according to the present invention, particularly a complete medium comprising all the components necessary to maintain and / or support in vitro cell growth, typically contains at least one or more sugar components, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffer components, one or more cofactors, and one or more nucleic acid components. They may additionally comprise chemically defined biochemicals such as recombinant proteins such as r-insulin, rBSA, r-transferrin, r-cytokines, etc.

[0035] The sugar components are all monosaccharides or disaccharides of the kind, such as glucose, galactose, ribose, or fructose (examples of monosaccharides), or sucrose, lactose, or maltose (examples of disaccharides).

[0036] Examples of amino acids according to the present invention include tyrosine, amino acids that make up proteins, particularly essential amino acids, leucine, isoleucine, lysine, methionine, phenylalanine, arginine, threonine, tryptophan, and valine, as well as non-protein amino acids of the type D-amino acids, thereby L-amino acids being preferred. The term amino acid further includes salts of amino acids of the type sodium salt, or their respective hydrates or hydrochloride salts. For example, tyrosine means L- or D-tyrosine, preferably L-tyrosine, and their salts, hydrates, or hydrochlorides.

[0037] Examples of vitamins include vitamin A (retinol, retinal, various retinoids, and four carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxamine, pyridoxal), vitamin B7 (biotin), vitamin B9 (folic acid, folinic acid), and vitamin B 12 These include vitamin C (cyanocobalamin, hydroxycobalamin, methylcobalamin), vitamin D (ergocalciferol, cholecalciferol), vitamin E (tocopherol, tocotrienol), and vitamin K (phylloquinone, menaquinone). Vitamin precursors are also included.

[0038] Examples of salts include components containing inorganic ions such as baicarbonate, calcium, chloride, magnesium, phosphate, potassium, and sodium, or trace elements such as Co, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Ni, Bi, V, and Zn. Examples include copper(II) sulfate pentahydrate (CuSO4.5H2O), sodium chloride (NaCl), calcium chloride (CaCl2.2H2O), potassium chloride (KCl), iron(II) sulfate, ammonium iron citrate (FAC), anhydrous sodium dihydrogen phosphate (NaH2PO4), anhydrous magnesium sulfate (MgSO4), anhydrous monohydrogen phosphate (Na2HPO4), magnesium chloride hexahydrate (MgCl26H2O), and zinc sulfate heptahydrate.

[0039] Examples of buffering agents include CO2 / HCO3 (carbonate), phosphate, HEPES, PIPES, ACES, BES, TES, MOPS, and TRIS. Examples of cofactors include thiamine derivatives, biotin, vitamin C, NAD / NADP, cobalamin, flavin mononucleotides and derivatives, glutathione, heme nucleotide phosphates, and derivatives.

[0040] The nucleic acid components according to the present invention are nucleic acid bases such as cytosine, guanine, adenine, thymine, or uracil; nucleosides such as cytidine, uridine, adenosine, guanosine, and thymidine; and nucleotides such as adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate.

[0041] Feed media may have various compositions compared to complete media. They typically contain amino acids, trace elements, and vitamins. They may also contain sugar components, however, sometimes for production reasons, sugar components are added to separate feeds.

[0042] Many biopharmaceutical production platforms are based on fed-batch cell culture protocols. The goal is typically to develop high-titer cell culture processes to reduce production costs in response to growing market demand. Besides the use of high-performance recombinant cell lines, improvements to cell culture media and process parameters are required to achieve maximum production capacity.

[0043] In a fed-batch process, the base medium supports initial growth and production, while the feed medium protects against nutrient depletion and sustains the production phase. The medium is selected to accommodate the different metabolic requirements between various production phases. Process parameter settings—including feed strategy and control parameters—define the optimal chemical and physical environment for cell growth and protein production.

[0044] In the perfusion process, cells are retained in the bioreactor through a cell retention device, while cell culture medium is continuously added and removed from the bioreactor via a pump. The advantages of perfusion are that the product can be removed from the bioreactor daily, thus reducing the exposure time of recombinant proteins to high temperatures and decreasing the redox potential or released cellular enzymes, thus potentially reaching extremely high cell densities (due to stable medium exchange) and potentially generating extremely fragile recombinant proteins.

[0045] The process for perfusion cell culture typically involves cultured cells in a bioreactor system that includes a bioreactor with a medium inlet and a harvest outlet, thereby i. Continuously, or one or more times, preferably continuously, during the cell culture process, fresh cell culture medium is injected into the bioreactor via the medium inlet. ii. Continuously during the cell culture process, or one or more times, preferably continuously, the harvest is removed from the bioreactor through the harvest outlet. The harvest typically includes cells, target products generated by the cells and liquid cell culture medium.

[0046] Poloxamers are amphiphilic polymers with two hydrophilic blocks and a central hydrophobic block. Poloxamers are polyethylene glycol (PEG) / polypropylene glycol (PPG) triblock copolymers, where one PPG block is flanked on both sides by PEG blocks. The polyethylene glycol (PEG) moiety is often also called the polyethylene oxide (PEO) moiety. The polypropylene glycol (PPG) moiety is often also called the polypropylene oxide (PPO) moiety.

[0047] Poloxamer (CAS number 9003-11-6) is typically used in cell culture applications, but is also called poloxamer 188 and has a general formula I, which preferably has x and z independently of 75-85 and preferably of 25-30. [ka] The poloxamer of the present invention can be found in the regions x=z=3 to 72 and y=5 to 12. Further information on poloxamers can be found in Hagers Handbuch der Pharmazeutischen Praxis, volume 9 "Stoffe PZ", 1994, pages 282 to 284.

[0048] Meloxapole has an inverted arrangement compared to poloxamers. This is why they are commonly called "inverted poloxamers." The central block of meloxapole is PEO (polyethylene oxide), sandwiched on both sides by PPO (polypropylene oxide) blocks (Schmolka 1977). [ka] The meloxapole reported in this invention can be found in the region where x=z=1~31 and y=13~173.

[0049] Poloxamer and Meloxapol share the same CAS number, 9003-11-6. The nomenclature for poloxamers and meloxapoles is similar and has been standardized in the aforementioned review by Schmolka (Schmolka 1977). Multiplying the first digit by 100 refers to the molecular weight of the PPO block, and multiplying the last digit by 10 gives the %EO of the molecule. To distinguish meloxapoles, an R is often added between the first and last digits to indicate its reverse structure.

[0050] Several poloxamers and meloxapoles are commercially available, with poloxamers being Pluronic® or Lutrol® (e.g., Pluronic® solutions, gels, or solids such as Pluronic® F-68) or meloxapole being Pluronic® R.

[0051] Alternatively, poloxamers and meloxapoles can be synthesized from raw materials according to methods known in the art (see, for example, U.S. Patent Nos. 3,036,118 and 3,740,421).

[0052] Table 1 summarizes exemplary meloxapoles. All listed meloxapoles are characterized by determining their polyethylene oxide percentage (%EO, wt / wt) and molecular weight distribution. %EO is, 1 The molecular weight distribution was determined using 1H NMR. The molecular weight distribution was determined by size exclusion chromatography (see, e.g., Method 1). Peak molecular weight (Mp) was used to characterize each polymer. [Table 1] Table 2 shows the same data for poloxamer 188 used as a reference in the example. [Table 2]

[0053] Detailed description of the invention The essence of this invention lies in the discovery that certain meloxapoles exhibit improved properties for use in cell culture. They not only provide shear protection, like poloxamer 188, but also show reduced foam formation. Suitable meloxapoles are also non-toxic to the cultured cells.

[0054] Poloxamer 188 is often added to cell culture media to protect against hydrodynamic stress caused by sparging and / or agitation. It reduces surface tension and, typically, increases foam formation. Since cells tend to adhere to the foam surface, they rise to the surface due to the foam, get trapped in the foam layer, and die. Here, it has been found that with various compositions and meloxapole, positive shear stress protection properties can be maintained, but foam formation is reduced.

[0055] As a result, the amount of antifoaming agent, such as antifoaming agent C, used to reduce foam formation can be reduced or, preferably, even eliminated when using cell culture media according to the present invention. In a preferred embodiment, the media according to the present invention contains less than half the amount of the most preferred non-antifoaming agent used, such as antifoaming agent C, in equivalent cell cultures under the same conditions and in the same media except that it does not contain meloxapole as defined in the present invention other than poloxamer 188. Typical antifoaming agents consist of oil (hydrocarbon or poly(dimethylsiloxane)), dispersed hydrophobic solid particles, or a mixture of both. Examples of antifoaming agents are preferably agents containing polydimethylsiloxane (PDMS) and optionally silica particles, such as Dow-Corning Q7-2587 (30.4% PDMS, 1.2-2.1% SiO2 particle O / W emulsion), Antifoam C from Sigma Aldrich (29.4% PDMS, 1.2-2-1% SiO2 particle O / W emulsion), and Foam Away from Gibco (30% simethicone emulsion in water), also known as dimethicone or simethicone, for example.

[0056] Preferably, the cell culture medium according to or used in the present invention does not contain poloxamer 188.

[0057] Meroxapol, which was identified as particularly suitable, has a peak molecular weight M between 1000 g / mol and 8000 g / mol, preferably between 1000 g / mol and 5000 g / mol. P , and meloxapole having a percentage of ethylene oxide between 55% and 95% (w / w).

[0058] In a preferred embodiment, peak molecular weight M P It is between 1800 g / mol and 3500 g / mol. In another preferred embodiment, the PEO percentage is between 55% and 80%. Most preferably, meloxapol has a peak molecular weight M between 800 g / mol and 3500 g / mol. P It has [specific characteristics], and the PEO percentage is between 55% and 80%.

[0059] Those skilled in the art know how to use meroxapol as an ingredient in cell culture media. They are also aware of the appropriate amounts and formats for use. Typically, meroxapol is applied to cell culture as part of the cell culture medium. However, it can also be applied separately. Meloxapole typically exists in the form of a solid, such as particles, or a liquid, such as an oil, or a highly viscous liquid like a paste, or an aqueous solution.

[0060] This invention relates to the peak molecular weight M between 1000 g / mol and 8000 g / mol. P It is a cell culture medium containing meroxapol with a percentage of ethylene oxide between 55 and 95% (w / w). In a preferred embodiment, meloxapol has a peak molecular weight M between 1800 g / mol and 3500 g / mol. P It has [specific characteristics], and the PEO percentage is between 55% and 95% (w / w).

[0061] In another preferred embodiment, the cell culture medium is free of defoaming agents or contains a reduced amount of defoaming agents, preferably less than 50% compared to the cell culture medium of the present invention that does not contain poloxamer 188. Preferably, the medium also does not contain poloxamer 188.

[0062] The concentration of meroxapole in the cell culture medium is preferably between 0.1 and 10 g / L, as calculated for liquid media. In a very preferred embodiment, the amount of meroxapole is between 0.5 and 2 g / L, as calculated for liquid media. Meroxapole may be one type of meroxapole as defined above, or a mixture of two or more types of meroxapole as defined above.

[0063] Preferably, the cell culture medium is a dry powder, dry granular medium, or liquid medium. In the case of a dry medium, the medium is dissolved in a suitable amount of water or aqueous buffer before use. The cell culture medium of the present invention can also be used for any type of cell culture. Cell culture is any setup in which cells are cultured.

[0064] Cell culture can also be carried out in any container suitable for cell culture, such as petri dishes, eyelets, bottles, tubes, wells, containers, bags, flasks, and / or tanks. Preferably, it is carried out in a bioreactor. Typically, the container is sterilized before use. Culture is typically carried out by incubation of cells in aqueous cell culture medium under suitable conditions such as suitable temperature, molar osmotic pressure, aeration, agitation, etc., which limits contamination by foreign microorganisms from the environment. Those skilled in the art recognize suitable incubation conditions for supporting or maintaining cell growth / culture.

[0065] The present invention thus relates to a process for cell culture, thereby enabling cells to achieve a peak molecular weight between 1000 g / mol and 8000 g / mol. P The cells are cultured in a liquid medium containing meloxapol with an ethylene oxide percentage between 55% and 95% (w / w).

[0066] Cell culture can be any setup suitable for the cultured cells. Preferably, it is batch, fed-batch, or perfusion cell culture. Preferably, the process for cultured cells includes the following steps: a) To provide a bioreactor b) Mixing the cells to be cultured with a cell culture medium according to the present invention. c) Incubate the mixture from step b). In a preferred embodiment, the process does not involve the addition of an antifoaming agent.

[0067] In one embodiment, the process includes the following steps: a) To provide a bioreactor b) Mixing the cells to be cultured with a cell culture medium according to the present invention. c) Incubate the mixture from step b), thereby adding the cell culture medium, in this case the feed medium, to the bioreactor for the entire duration of the cell incubation time in step c), or continuously once or several times. The feed medium may be a cell culture medium according to the present invention, but it may also be a feed medium that does not contain meroxapol. Preferably, it does not contain meroxapol.

[0068] In one embodiment, the bioreactor is a perfusion bioreactor. A perfusion bioreactor is a bioreactor on which perfusion cell culture can be performed. It typically includes a sealed bioreactor vessel, a stirrer in the vessel, a line for introducing fresh medium, a harvest line for removing a harvest stream containing cells, liquid medium, and target product from the bioreactor, and a cell holding device in the harvest line that holds the cells while the liquid portion of the harvest can be collected. A review on perfusion cell culture providing details on a preferred setup can be found in "Perfusion mammalian cell culture for recombinant protein manufacturing - A critical review" Jean-Marc Bielser et al., Biotechnology Advances 36 (2018) 1328-1340.

[0069] In the perfusion process, cells are retained in the bioreactor through a cell retention device, while cell culture medium is continuously added and removed from the bioreactor via a pump. The advantages of perfusion are that the product can be removed from the bioreactor daily, thus reducing the exposure time of recombinant proteins to high temperatures and decreasing the redox potential or released cellular proteases, thus potentially allowing for extremely high cell densities (due to stable medium exchange) and potentially generating extremely fragile recombinant proteins.

[0070] In one embodiment, the process of the present invention comprises cultured cells in a bioreactor system including a bioreactor with a culture medium inlet and a harvest outlet, thereby i. Continuously, or one or more times, preferably continuously, during the cell culture process, a new cell culture medium according to the present invention is injected into the bioreactor via the medium inlet. ii. Continuously during the cell culture process, or one or more times, preferably continuously, the harvest is removed from the bioreactor through the harvest outlet. The harvest typically includes cells, target products generated by the cells and liquid cell culture medium.

[0071] When using the cell culture medium according to the present invention, the culture exhibits equal productivity and reduced foam formation compared to a cell culture performed under the same conditions, except that a medium containing poloxamer 188 is used instead of meloxapole as defined above.

[0072] The present invention also relates to a method for reducing foam formation in agitated and / or spagged cell cultures, thereby reducing the peak molecular weight of the cells between 1000 g / mol and 8000 g / mol. P The cells were cultured in a liquid medium containing meloxapol with an ethylene oxide percentage between 55% and 95% (w / w), and the peak molecular weight M was between 1000 g / mol and 8000 g / mol. PCompared to cells cultured under the same conditions in a cell culture medium containing poloxamer 188 instead of meloxapol with an ethylene oxide percentage between 55% and 95% (w / w), foam formation is reduced. Preferably, no defoaming agent is used in the method for suppressing foam formation.

[0073] The present invention also relates to a method for culturing cells in a stirred and / or spaged cell culture, thereby enabling cells to reach a peak molecular weight between 1000 g / mol and 8000 g / mol. P The cells were cultured in a liquid medium containing meloxapol with an ethylene oxide percentage between 55% and 95% (w / w), and the peak molecular weight M was between 1000 g / mol and 8000 g / mol. P Compared to cells cultured under the same conditions in a cell culture medium containing poloxamer 188 instead of meloxapol with an ethylene oxide percentage between 55% and 95% (w / w), the amount of defoamer in the liquid medium was reduced. Preferably, the amount of defoamer is reduced by at least 25%, preferably at least 50%. Most preferably, no defoamer is used in the culture method of stirred culture and / or sparged cultured cells.

[0074] The culture medium and method of the present invention, for the first time, provide shear stress protection on the one hand, and on the other hand, the possibility of avoiding complications typically associated with foam stabilization and the resulting need for additional defoaming agents. Shear stress protection is provided without adversely affecting foam formation and stabilization. It is even possible to increase the amount of meloxapole in the cell culture medium without adversely affecting foam formation. This provides cell culture experts with the flexibility to set ideal culture conditions.

[0075] These results were unexpected. For example, poloxamer 188 and meloxapole 12R8 had peak molecular weights (M pAlthough the molecular weights of the %EO and PPO blocks, as well as the surface tension values ​​of the solutions in the cell culture medium, are similar, they nevertheless exhibit very different foam behavior. As seen in Example 2, the meloxapol 12R8 solution produced foam that did not reach the top of the foam analyzer column and eventually equilibrated to a plateau height between 5 cm and 6 cm. In contrast, the sample to which poloxamer 188 was added reached the maximum foam height of the analyzer column in a very short time.

[0076] All applications, patents, and publications cited above and below, as well as the full disclosure of the corresponding patent application EP22168244.6 filed on April 13, 2022, are incorporated herein by reference.

[0077] example Method 1 All size exclusion chromatography (SEC) measurements are performed as follows: Calibration standard: PEG (Mp: 430, 982, 1,960, 3,020, 6,690, 12,300, 26,100 and 44,000 g / mol) Eluent:THF Flow rate: 1mL / min Injection volume: 100μl Column: Particle size = 5 μm, Material = Styrene-divinylbenzene Temperature: 40℃ Detector: Refractive index (RI)

[0078] Molecular weight distribution curves were measured for all meloxapoles, and the peak value (Mp) was determined (Tables 1 and 2). An exemplary distribution curve is shown in Figure 13, which shows the molecular weight distribution determined by size exclusion chromatography for meloxapole 8R6. Mp indicates the peak molecular weight of the specific meloxapole.

[0079] Method 2: Measurement of foam generation in meloxapol solution in cell culture medium at 37°C Preparation of meroxapole solution in cell culture medium A solution of the target meloxapole in Cellvento® 4CHO cell culture medium without poloxamer 188 (Merck KGaA) was prepared at a selected concentration and then stirred or mixed through a roller mixer for at least 2 hours until complete dispersion was achieved. The solution was prepared by weight. The cell culture medium was prepared according to the manufacturer's instructions.

[0080] bubble measurement Foam generation was tested using a Dynamic Foam Analyzer DFA 100 (Kruess GmbH). The following parameters were adopted: Temperature: 37℃ Foaming method: Volume control (air) Flow rate: 0.3L / min Liquid capacity: 15mL Column CY4575-40mm t.prism Filter FL4551 - Paper, 12-25 μm, [ka] Height illumination, blue - λ = 469 nm Sample holder SH4511 sparging Camera height: 60mm Camera position: 2 Structural lighting: 30% Height lighting: 20%

[0081] The solution was injected into the glass column of the Foam Analyzer DFA 100. The solution was heated to 37°C, which is the temperature used in bioreactors.

[0082] The gas flow was initiated, and foam formation (foam height, liquid height, and foam structure) was recorded using a Foam Analyzer.

[0083] If the foam reached the maximum column height (210 mm), the gas flow automatically stopped to prevent overflow, and the foam decay was monitored for a selected time without gas sparging. If the foam did not reach the top of the column, the gas continued to flow for a selected time (typically 60 minutes), then stopped, and the foam decay was monitored.

[0084] Method 3: Measurement of static surface tension Static surface tension was measured using a force tensiometer K100C (Kruss GmbH). A Wilhelmie plate was used for the measurement.

[0085] The following parameters were adopted. Measurement temperature: 37°C Measurement time: 300s Data points: 200 Frequency: 1Hz Data is acquired until the standard deviation of the measured surface tension is less than 0.07 mN / m from the average value of 200 data points.

[0086] The solution was poured into the glass vessel of the Force Tensiometer K100C. Since this temperature is used in bioreactors, the solution was heated to 37°C. Immediately before starting the measurement, the Wilhelmie plate was heated with a flame until it glowed red to remove any contaminants and ensure a zero contact angle.

[0087] Method 4 Fed-batch cell culture in rotating tubes The protective capacity of meloxapol during fed-batch cell culture was evaluated. CHOK1GS cells were used. Culture was performed in a rotating tube (TPP, Art. No. 87050) with a final volume of 30 mL at 37°C, in a humidified atmosphere of 5% CO2, and at a rotation speed of 320 rpm (incubator deflection 25 mm).

[0088] Prior to the start of the feeding process, cells were cultured in Cellvento® 4CHO (Merck KGaA, 103795) cell culture medium containing 2 g / L poloxamer 188. HT-supplements 100x (sodium hypoxanthine (10 mM) and thymidine (1.6 mM), Gibco®, Life Technologies, Art. No. 11067-030) and L-methionine sulfoximine (Sigma Aldrich M5379) were added to this medium. For each condition, Cellvento® 4CHO cell culture medium without poloxamer 188 (Merck KGaA) was used, and the selected meloxapole was added to achieve a concentration of 1 g / L. The solution was stirred for at least 2 hours to ensure complete dispersion.

[0089] On day 0 of the feeding culture, the cells were centrifuged at 2000 rpm for 5 minutes, then resuspended in 4CHO+HT without poloxamer 188 and pooled. CHOK1 GS cells under different poloxamer conditions (4 replicates each) showed 0.2-10 6 Cells were seeded at a concentration of cells / mL. The cells were fed using a feed solution.

[0090] Glucose concentration was measured daily, starting from day 3. Continuous glucose feeding was maintained throughout the entire fermentation process as needed. The culture was carried out for 17 days.

[0091] VCD and survival rate analysis using ViCell (Beckman Coulter), and IgG determination using Cedex (Bio HT Analyzer, Roche) were performed daily.

[0092] Fed-batch cell culture in a bioreactor To evaluate the protective and low-foaming properties of meloxapol in CHOK1 GS cell culture, fed-batch cell culture was performed in a bioreactor.

[0093] Glass bioreactors (Eppendorf, DASGIP® Parallel bioreactor system Catalog. 76DG08CC and 76DG04CCBB) were used. The bioreactors were equipped with an L-sparger and a pitched-blade impeller. A foam trap was added to prevent foam overflow.

[0094] Prior to the start of the feed batch, cells were cultured and adapted in meloxapole and poloxamer 188 solutions in Cellvento® 4CHO (formulation without poloxamer, Merck KGaA, 4.74000.9999), respectively. The concentration was 2 g / L. HT-Supplement 100x (sodium hypoxanthine (10 mM) and thymidine (1.6 mM), Gibco®, Life Technologies, Art. 11067-030) and L-methionine sulfoximine (Sigma Aldrich M5379) were added to this medium.

[0095] The feed batch was performed according to the following parameters. [Table 3] 0.2–0.4% of the antifoaming agent C was added only when foam began to be collected in the foam trap. This phenomenon occurred only with poloxamer 188. No significant foam formation occurred with all other meloxapols tested, and therefore, the addition of antifoaming agent C was unnecessary.

[0096] Example 1: Foam Analysis The foam generation behavior of the selected meloxapole was investigated. The experimental setup and procedure are described in Method 2.

[0097] Foam stabilized with meloxapol was compared to foam stabilized with poloxamer 188, a benchmark for animal-free cell culture media. Different solutions were prepared. Meloxapol was added to Cellvento® 4CHO cell culture medium (without poloxamer 188) at concentrations of 1000 ppm (1 g / L) and 2000 ppm (2 g / L).

[0098] The foam generation plots for the medium with a meloxapole concentration of 1000 ppm are reported in Figure 1, and for the medium with a meloxapole concentration of 2000 ppm, they are reported in Figure 2.

[0099] At both concentrations, the bubbles stabilized by poloxamer 188 reached a maximum bubble height of 210 mm (i.e., the length of the bubble analyzer column) within 50 seconds. At that point, the instrument automatically stopped the measurement to prevent overflow.

[0100] Meloxapol exhibited significantly different behavior. Foam stabilized by meloxapol did not reach the top of the foam analyzer column, and air sparging continued throughout the measurement time. The foam grew rapidly in the first few seconds of gas sparging, reaching a maximum height between 30–69 mm for the 1000 ppm solution and between 24–162 mm for the 2000 ppm solution. Subsequently, the foam height rapidly decreased, stabilizing at a constant height between 24–51 mm for the 1000 ppm solution and between 24–63 mm for the 2000 ppm solution (see Figures 1, 2, 3, and 4).

[0101] The decrease in foam height and the formation of a constant foam level, i.e., plateau height, can be explained in terms of the equilibrium between foam formation due to sparging and foam collapse due to the combination of wastewater and foam.

[0102] Despite meloxapol being less effective at stabilizing foam compared to poloxamer 188, significant differences in foam stabilization ability were observed for different meloxapols. Meloxapol 9R8 and 12R8 were more effective at stabilizing foam and resulted in higher foam heights compared to other meloxapols. In particular, meloxapol 8R6 represented the highest maximum foam height at the lowest foam plateau at both concentrations.

[0103] Poloxamer 188 and meloxapole 9R8 exhibit extremely different foam behavior. Foam stabilized with poloxamer 188 reaches a maximum foam height of 21 cm within 1 minute, while foam with meloxapole 9R8, at both concentrations, has a maximum foam height of approximately 7 cm and reaches equilibrium at plateau heights of around 5 cm at 1000 ppm and 6 cm at 2000 ppm.

[0104] Another surprising observation was that the average maximum bubble height and plateau height remained similar even when the concentration was doubled. Only for meloxapol 12R8 was the average maximum bubble height significantly higher at 2000 ppm compared to 1000 ppm.

[0105] These unexpected foam analysis data could be explained by static surface tension measurements performed at 37°C using the same solution immediately prior to the foam analysis. Increasing the concentration did not lead to a significant decrease in surface tension, with differences of 1–2.5 mN / m. The largest difference in surface tension at doubling the concentration was measured for the meloxapol 12R8 solution (i.e., 4 mN / M), and this was the same sample that also showed the largest difference in average foam height.

[0106] No significant correlation was found between the surface tension values ​​of each sample and the average plateau height or average maximum bubble height. In principle, lower surface tension leads to greater stabilization of the interface region, and therefore to more bubbles and more foam. However, the meloxapol 8R6 solution, which has lower surface tension compared to the other materials ((48.7±0.2) mN / m at 1000 ppm, (46.5±0.3) mN / m at 2000 ppm), still results in the lowest bubble height.

[0107] Poloxamer 188 and meloxapole 12R8 have similar peak molecular weight (Mp), %EO, PPO block molecular weight, and surface tension values ​​of the solution in cell culture medium; however, they exhibit very different foam behavior. The meloxapole 12R8 solution produced foam that did not reach the top of the foam analyzer column, and it subsequently equilibrated at a plateau height between 5 cm and 6 cm.

[0108] Table 3 shows the static surface tension of meloxapole solution in cell culture medium at concentrations of 1000 ppm and 2000 ppm. The reported data are the mean and standard deviation of repeated measurements at 37°C. [Table 4]

[0109] Example 2: Use of meloxapole in fed-batch culture Rotating tube culture Fed-batch cell culture (see Method 4) was performed, and the shear stress protection properties of various meloxapoles were investigated in comparison to the benchmark poloxamer 188.

[0110] As observed in the foam analysis data, meloxapol resulted in significantly lower foam heights compared to poloxamer 188, making it extremely attractive as a shear stress protectant for cell culture.

[0111] CHOK1 GS cells were cultured in a fed-batch container in a rotating tube for 17 days according to the procedure described in Method 4. All meloxapole solutions were tested in Cellvento® 4CHO cell culture medium without poloxamer 188. Each solution had a concentration of 1 g / L meloxapole (i.e., 1000 ppm).

[0112] As shown in Figures 5, 6, and 7, the viability, viable cell density, and IgG production of cells cultured with meloxapole were comparable to those of cells cultured with poloxamer 188, even when the concentration of 188 was doubled (2 g / L).

[0113] Figure 6 more clearly illustrates the differences between the different samples. Up to day 7, all samples had comparable VCD within the margin of error. After day 10 of culture, cells cultured with both concentrations of poloxamer 188 began to decline, while cells cultured with the other meloxapole continued to proliferate until day 13. Antibody production from cells cultured with meloxapole, particularly 8R7 and 8R6, is comparable to that from cells cultured with poloxamer 188.

[0114] This experiment demonstrated that the described meloxapol is an effective shear stress protectant for fed-batch culture in rotating tubes, and that antibody productivity is comparable to that of culture using poloxamer 188. Observing how well meloxapol supports cell proliferation is remarkable, especially considering that poloxamer 188 is essential for culture, even under low shear stress conditions such as fed-batch culture in rotating tubes. The essential role of poloxamer 188 is highlighted in Figure 8, where cell viability in culture without any poloxamer is compared to conditions including 1 g / L poloxamer 188. Either poloxamer 188 or a low-foaming alternative such as meloxapol as described in this invention is necessary to achieve cell proliferation in suspension culture.

[0115] Culturing in rotating tubes, due to its setup (no sparging and therefore no foam formation), does not allow for comparison of foam formation under different conditions. Evaluation of shear stress protection combined with low foaming capacity will be performed in bioreactor cultures.

[0116] Bioreactor culture Fed-batch cell culture in a bioreactor was performed to simultaneously evaluate the shear stress protection and foam stabilization properties of different meloxapoles compared to the benchmark poloxamer 188. Culture in a bioreactor, in contrast to rotating tube culture, involves air sparging and mechanical agitation, which could lead to foam formation. Therefore, a bioreactor is suitable for evaluating the performance of meloxapoles.

[0117] CHOK1 GS cells were cultured in a fed-batch container in a glass bioreactor for 14 days according to the procedure described in Method 4. All meloxapol was tested in Cellvento® 4CHO cell culture medium without poloxamer 188. Each condition had a concentration of 2 g / L (i.e., 2000 ppm) of meloxapol or poloxamer 188.

[0118] As shown in Figures 9, 10, and 11, the viability, viable cell density, and IgG production of cells cultured in meloxapole 8R7 and 8R6 are comparable to the data for cells cultured in poloxamer 188. Cells cultured in meloxapole 12R8 exhibit lower VCD and IgG titers, but still show comparable viability. This observation may hint that meloxapole 12R8 is a less efficient shear stress protectant compared to other conditions. This culture setup with increased shear stress allowed us to more deeply evaluate the protective performance and distinguish meloxapole. Antibody production in cells cultured in meloxapole 8R7 and 8R6 is comparable to that of cells cultured in poloxamer 188.

[0119] The difference in foam stabilized with meloxapol and poloxamer 188 is significant. Foam formed in bioreactors containing meloxapol was extremely minimal throughout the culture. The maximum foam level was recorded at the end of the culture on day 14: 150 mL for meloxapol 8R7, 50 mL for meloxapol 8R6, and none for meloxapol 12R8.

[0120] Instead, poloxamer 188 reached a foam level of 800 mL in just 7 days, requiring defoamer C to avoid foam overflow. Without the addition of defoamer C, the overflow could have led to overpressure, leakage, contamination, and ultimately, the interruption of the experiment. Starting on day 10, defoamer C was added daily to the three bioreactors containing poloxamer 188. Nevertheless, the foam became increasingly stable. Cultures supported with meloxapol did not require defoamer C, and they also did not pose a risk of foam breakdown.

[0121] The use of meloxapol 8R7 and 8R6 has been demonstrated to yield cell proliferation and antibody productivity comparable to poloxamer 188, while simultaneously significantly reducing foam formation and avoiding the need for antifoaming agent C. The use of selected meloxapols may be highly beneficial in bioprocesses, reducing risks and avoiding problems associated with the addition of antifoaming agents.

Claims

1. Peak molecular weight M between 1000 g / mol and 8000 g / mol ｐ A cell culture medium comprising meloxapol having a percentage of polyethylene oxide between 55% (weight / weight) and 95% (weight / weight).

2. Peak molecular weight M ｐ The cell culture medium according to claim 1, characterized in that the concentration is between 1000 g / mol and 5000 g / mol.

3. The cell culture medium according to claim 1, characterized in that the percentage of polyethylene oxide is between 55% and 80%.

4. The cell culture medium according to claim 1, characterized by containing meloxapol in an amount between 0.1 and 10 g / L calculated for the liquid medium.

5. The cell culture medium according to claim 1, characterized in that it is a chemically defined culture medium.

6. The cell culture medium according to claim 1, characterized in that it is a dried powder or a dried compressed culture medium.

7. The cell culture medium according to claim 1, characterized in that the cell culture medium contains at least one sugar component, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffering components, one or more coexisting factors, and one or more nucleic acid components.

8. The cell culture medium according to claim 1, characterized in that it does not contain an antifoaming agent.

9. A process for cell culture, thereby raising the cell's peak molecular weight between 1000 g / mol and 8000 g / mol ｐ The process involves culturing in a liquid medium containing meloxapol having a percentage of polyethylene oxide between 55% (weight / weight) and 95% (weight / weight).

10. The process for cell culture according to claim 9, characterized in that the process includes stirring and / or sparging.

11. Peak molecular weight M ｐ The cell culture process according to claim 9, characterized in that the concentration is between 1000 g / mol and 5000 g / mol.

12. The cell culture process according to claim 9, characterized in that the percentage of polyethylene oxide is between 55% and 80%.

13. The cell culture process according to claim 9, characterized in that the amount of antifoaming agent in the liquid medium is reduced compared to a cell culture medium containing 188-poloxamer instead of meloxapol, except that the other components are the same.

14. The cell culture process according to claim 9, characterized in that the liquid culture medium does not contain an antifoaming agent.

15. A method for reducing foam formation in stirred and / or spaged cell cultures, wherein the cells have a peak molecular weight between 1000 g / mol and 8000 g / mol. ｐ The cells were cultured in a liquid medium containing meloxapol with a percentage of polyethylene oxide between 55% (g / mW) and 95% (g / mW), and the peak molecular weight M was between 1000 g / mol and 8000 g / mol. ｐ The method wherein foam formation is reduced compared to cells cultured under the same conditions in a cell culture medium containing poloxamer 188 instead of meloxapol, which has polyethylene oxide in a percentage between 55% (weight / weight) and 95% (weight / weight).

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