Medium comprising keto acid

Abstract

The present invention relates to a culture medium, preferably a cell culture medium, comprising at least one ketoacid selected from the group consisting of ketoleucine, ketovaline, ketoisoleucine and ketophenylalanine and / or salts of these ketoacids. The invention further relates to the use of the culture medium according to the invention for culturing cells, preferably plant cells, animal cells or mammalian cells. Another aspect of the invention relates to a method of manufacturing a cell culture product comprising the steps of: (i) providing cells capable of producing said cell culture product; (ii) contacting the cells with a culture medium according to the invention; and (iii) obtaining the cell culture product from the culture medium or from the cells.

Description

[0001] This application is a divisional application of CN201980074128.3. Field of the Invention

[0002] The present invention relates to biotechnological production methods. More specifically, the present invention relates to improved culture media for use in biotechnological production methods, methods using such improved culture media, and products obtained from methods using the improved culture media. Background of the Invention

[0004] Biotechnological methods are widely used to produce biological products. These methods generally involve culturing cells in a culture medium under conditions that allow growth of the cultured cells and product formation. Cells that can be used for biotechnological production are bacterial cells, fungal cells, yeast cells, and cells of animal or plant origin.

[0005] Animal cell culture has long been used to produce biological products, such as therapeutic proteins, polypeptides, oligopeptides or other biomolecules, such as therapeutic polysaccharides. In such cell culture processes, animal cells, which are usually genetically modified to produce the desired product, are cultured in liquid, solid or semi-solid medium for cell proliferation and product formation. A significant advantage of cell culture is that animal and plant cells are able to perform post-translational modifications on primary products, such as folding and post-translational modifications of polypeptides.

[0006] In biotechnology production methods, especially in animal cell culture, the control and optimization of cell culture conditions are crucial for cell proliferation and product formation. A decisive factor is the culture medium composition. The concentration and quality of the final cell culture product depend to a large extent on the culture medium composition. Animal and plant cell culture are particularly in need in terms of the required nutritional composition and culture conditions. The required nutrients include not only the basic sources of carbon, nitrogen and energy, such as sugars and ammonia, but also more complex nutrients, such as essential amino acids, growth factors and vitamins. For this reason, supplementing animal cell culture medium with complex nutrients such as serum has been used to provide animal cells with a wide variety of nutrients. However, regulatory and safety issues and problems related to the heterogeneity of available serum sources have led to industry efforts to eliminate serum and other culture media with uncertain components from industrial cell culture processes. However, cell cultures grown in serum-free culture media generally show nutritional deficiencies. For this reason, a lot of effort has been made to identify those nutrients of complex culture media and their optimal concentrations, which are necessary for satisfactory growth and protein formation in cell cultures. A common strategy to overcome nutritional deficiencies is to feed the spent culture medium components during the culture process. The highly concentrated solution used for replenishment is called feed medium. Optimizing their composition follows the same logic as for basal media.

[0007] A specific subset of applications is the production of viruses as vaccines or as vectors for gene therapy. In both cases, the cell culture process shows a clear separation into two phases. The first phase is the manufacture of the virus, aiming to maximize the virus yield. The second phase is the transfection of the cells with the vector. Here, the culture medium ideally supports high transfection efficiency.

[0008] Providing amino acids to cell cultures is known to have a significant effect on growth rate and yield. Glutamine is routinely used in cell culture media and feeds because it is an important source of carbon, nitrogen and energy for cultured cells. It has been shown that the amount of glutamine required for optimal growth of animal cell cultures is 3 to 10 times the amount of other amino acids (Eagle et al., Science 130: 432-37). However, glutamine is unstable as a nutrient when dissolved in water at elevated temperatures (e.g., heat sterilization conditions) because pyroglutamic acid and ammonia are formed under heating (Roth et al., 1988, In VitroCellular & Developmental Biology 24(7): 696-98). Therefore, glutamic acid is usually used instead of glutamine in cell culture media (Cell Culture Technology for Pharmaceutical and Cell-basedTherapies, 52, Sadettin et al., ed., Taylor and Francis Group, 2006).

[0009] The use of α-ketoglutarate (AKG), a keto derivative of glutaric acid, which plays a key role in both L-glutamine metabolism and the Krebs energy cycle, in cell culture applications has been described previously (Hassel & Butler, Journal of Cell Science 96, 501-508, Evonik Nutrition & Care GmbH, Scientific overview: cQrex™ AKG, February 2016). It could be demonstrated that in the case of CHO and hybridoma cell lines, by adding AKG to commercially available culture media, the maximum viable cell density (VCD) could be increased by more than 30% and the overall viable cell density (IVCD) could be increased by more than 50%.

[0010] Although the cell culture media described in the above-mentioned prior art provide satisfactory performance and characteristics for certain cell culture processes, the culture media of the prior art are still not optimal. There is still a need for further improved culture media that promote better growth and product formation in biotechnological production processes. This is particularly relevant for the emerging field of cell-based virus production, whether as a vaccine or for treatment. In general, the key challenge in this field is to remove serum to improve proliferation performance and increase viral differentiation rate. The latter is particularly relevant for these two applications. In the case of an epidemic, large amounts of vaccines need to be made in a very short time. For gene therapy, minimizing the time between starting vector production and administration is a key parameter to ensure the success of the treatment.

[0011] It is therefore an object of the present invention to provide an advanced culture medium which promotes better growth and product formation of various cell lines and additionally reduces the amount of toxic ammonia. SUMMARY OF THE INVENTION

[0013] The present invention solves the above-mentioned disadvantages of known culture media. The present invention is defined by the terms of the attached independent claims. Preferred embodiments of the present invention are defined by the dependent claims.

[0014] Therefore, the present invention relates to a culture medium, preferably a cell culture medium, comprising at least one ketoacid selected from the group consisting of ketoleucine, ketovaline, ketoisoleucine and ketophenylalanine and / or salts of these ketoacids.

[0015] The present invention further relates to the use of the culture medium according to the invention for culturing cells, preferably plant cells, animal cells or mammalian cells.

[0016] Another aspect of the present invention relates to a method for producing a cell culture product, comprising the steps of: (i) providing cells capable of producing the cell culture product; (ii) contacting the cells with a culture medium according to the present invention; and (iii) obtaining the cell culture product from the culture medium or the cells.

[0017] Preferred embodiments of the present invention are described in further detail in the following detailed description of the invention.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Viral vector titers of adeno-associated virus (AVV) type 9 (AAV9) and adeno-associated virus type 2 (AAV2) in human embryonic kidney (HEK) cells in the absence and presence of a mixture of branched-chain keto acid (BCKA) potassium salts according to the invention are described.

[0020] Figure 2The infectivity of viral vectors (measured by luciferase activity) of adeno-associated virus (AVV) type 9 (AAV9) and adeno-associated virus type 2 (AAV2) in HEK cells in the absence and presence of a mixture of potassium salts of branched-chain keto acids (BCKAs) according to the present invention is described.

[0021] Figure 3 Depicted are the overall viable cell densities of the third passage of Chinese hamster ovary (CHO) cells cultured in cell culture medium containing different amounts of keto-leucine instead of L-leucine.

[0022] Figure 4 Shown are the relative concentrations of antibodies produced by CHO cells cultured in cell culture medium containing varying amounts of keto-leucine instead of L-leucine.

[0023] Figure 5 Shown are the maximum viable cell densities of CHO cells cultured in cell culture media comprising different amounts of ketovaline in addition to L-valine.

[0024] Figure 6 Shown are the relative concentrations of antibodies produced by CHO cells cultured in cell culture medium containing different amounts of ketovaline in addition to L-valine. DETAILED DESCRIPTION OF THE INVENTION

[0026] α-Keto acids have different functions in metabolism. Keto acid analogs of branched-chain amino acids play an important role in amino acid metabolism, especially in skeletal muscle and liver. One third of muscle protein consists of branched-chain amino acids that cannot be formed by the body but must be taken up with the diet. In the muscles, especially in the case of physical exercise, protein is constantly synthesized and broken down, wherein during the breakdown of amino acids the corresponding α-keto acids are formed by transferring the amino group to a carrier. The resulting keto acids can then be further enzymatically oxidized for energy production. The carrier is transported to the liver, where it releases ammonia, which is converted into urea and excreted via the kidneys.

[0027] It is already known that α-keto acids derived from branched-chain amino acids are used for pharmaceutical purposes. For example, α-ketoisocaproate (ketoleucine) is particularly useful for reducing protein breakdown in muscle, and for reducing urea formation caused by protein breakdown after muscle surgery (US 4,677,121). The use of ketoleucine in malnutrition, muscular dystrophy or uremia and other diseases, which are secondary results of protein breakdown in muscle, has also been described. Ketoleucine is administered intravenously in such cases. In addition, it has been proposed to administer α-keto acids of leucine, isoleucine and valine to patients who must maintain a protein-reduced diet (e.g., due to renal failure) (US 4,100,161). The role of α-keto acids in protein metabolism is also described in Walser, M. et al., Kidney International, Vol. 38 (1990), pp. 595-604, regarding various medical indications.

[0028] In contrast, in the field of functional foods, branched-chain amino acids are used directly to support muscle accumulation, for example in athletes (Shimomura, Y. et al., American Society for Nutrition). The use of α-keto acids of leucine, isoleucine and valine for improving muscle performance and also for supporting muscle recovery after fatigue is described in US 6,100,287, wherein salts of the corresponding anionic keto acids with cationic amino acids, such as arginine or lysine, are used as counterions. However, as a result, polyamines are also formed, which are known to cause apoptosis (programmed cell death). The excretion of the decomposition products of the polyamines takes place through the kidneys, which are therefore further stressed.

[0029] However, no effect has been shown in cultured cells so far.

[0030] According to the present invention, "medium" is understood to be a liquid or solid medium containing nutrients, which is suitable for nourishing and supporting the life and / or product formation of cells in the culture. According to the present invention, the cells cultured can be bacterial cells, yeast cells, fungal cells, animal cells such as mammalian cells or insect cells and / or plant cells such as algae. Usually, the culture medium provides the essential and non-essential amino acids, vitamins, at least one energy source, lipids and trace elements required for the cell to maintain life, growth and / or product formation. The culture medium can also be included in the components that promote growth and / or survival above the minimum rate, including hormones and growth factors. The culture medium preferably has pH and salt concentrations that support the life, growth and / or product formation of the cells. The culture medium according to the present invention preferably contains all nutrients necessary for the life and proliferation of the cell culture. The culture medium can be a defined or undefined composition. A preferred culture medium is a defined composition culture medium.

[0031] According to the present invention, a "defined medium" is a medium that does not contain cell extracts, cell hydrolysates or protein hydrolysates. Preferred defined media do not contain components of unknown composition. As generally understood by those skilled in the art, defined media generally do not contain components of animal origin. Preferably, all components of a defined medium have known chemical structures. A preferred defined medium is a serum-free medium. Other preferred defined media are synthetic media. Other media except "defined media" are referred to as "complex" media.

[0032] "Cell culture medium" is to be understood as a culture medium which is suitable for maintaining the life, proliferation and / or product formation of animal cells and / or plant cells. In certain embodiments, a distinction can be made between a basal culture medium and a feed culture medium.

[0033] "Basal medium" should be understood to be a solution or substance containing nutrients, in which the culture of cells is started. "Feed medium" should be understood to be a solution or substance with which cells are fed after the culture process begins. In certain embodiments, the feed medium contains one or more components that are not present in the basal medium. The feed medium can also lack one or more components present in the basal medium. Preferably, the concentration of nutrients in the feed medium exceeds the concentration in the basal medium to avoid loss of productivity due to dilution.

[0034] According to the present invention, "nutrients" are compounds or substances that are required for cell survival and growth. Nutrients are preferably absorbed by cells from the environment. Nutrients can be "organic nutrients" and "inorganic nutrients". Organic nutrients include carbohydrates, fats, proteins (or their building blocks, such as amino acids) and vitamins. Inorganic nutrients are inorganic compounds, such as dietary minerals and trace elements. "Essential nutrients" are nutrients that cells cannot synthesize themselves and therefore must be provided to the cells by the culture medium.

[0035] According to the present invention, "cell culture product" is understood to be any useful biological compound produced by cells in cell culture. Preferred cell culture products of the present invention are therapeutic proteins, diagnostic proteins, therapeutic polysaccharides such as heparin, antibodies such as monoclonal antibodies, growth factors, interleukins, peptide hormones, enzymes, and viruses including but not limited to viral vectors and vaccines. In other embodiments (including but not limited to the cultivation of stem cells for therapeutic use), the cells themselves are understood to be cell culture products.

[0036] In the context of the present invention, "amino acid" is understood to be a molecule comprising an amino functional group (-NH2) and a carboxylic acid functional group (-COOH) as well as side chains specific to the respective amino acid. In the context of the present invention, α- and β-amino acids are included. The preferred amino acids of the present invention are α-amino acids, in particular the following 20 "natural amino acids":

[0037] Alanine (Ala / A)

[0038] Arginine (Arg / R)

[0039] Asparagine (Asn / N)

[0040] Aspartic acid (Asp / D)

[0041] Cysteine ​​(Cys / C)

[0042] Glutamate (Glu / E)

[0043] Glutamine (Gln / Q)

[0044] Glycine (Gly / G)

[0045] Histidine (His / H)

[0046] Isoleucine (Ile / I)

[0047] Leucine (Leu / L)

[0048] Lysine (Lys / K)

[0049] Methionine (Met / M)

[0050] Phenylalanine (Phe / F)

[0051] Proline (Pro / P)

[0052] Serine (Ser / S)

[0053] Threonine (Thr / T)

[0054] Tryptophan (Trp / W)

[0055] Tyrosine (Tyr / Y)

[0056] Valine (Val / V)

[0057] In the context of the present invention, the expression "natural amino acid" is understood to include the L-form and the D-form of the 20 amino acids listed above. However, the L-form is preferred. In one embodiment, the term "amino acid" also includes analogs or derivatives of those amino acids.

[0058] According to the invention, a "free amino acid" is understood to be an amino acid having its amino group and its (α-) carboxyl functional group in free form, i.e. not covalently bonded to other molecules, e.g. not forming a peptide bond. A free amino acid may also be present in the form of a salt or a hydrate. When an amino acid is referred to as part of or in an oligopeptide, this is understood to mean that part of the corresponding oligopeptide structure is derived from the corresponding amino acid according to the known mechanisms of biochemistry and peptide biosynthesis.

[0059] According to the invention, "branched chain amino acids" (BCAA) are understood to be amino acids having aliphatic side chains (bonded to a central carbon atom of three or more carbon atoms) with branches. Among the proteinogenic amino acids, there are three BCAAs: leucine, isoleucine and valine. Non-proteinogenic BCAAs include 2-aminoisobutyric acid.

[0060] According to the invention, "keto acid" is understood to be an organic compound which comprises a carboxylic acid group and a keto group.

[0061] "α-keto acids" are understood to be keto acids having a keto group adjacent to a carboxylic acid. The keto acids according to the invention are selected from the group consisting of keto analogs of amino acids, i.e. ketoleucine, ketovaline, ketoisoleucine and ketophenylalanine and / or salts of these keto acids. Salts of keto acids are also intended to include mixed salts of such keto acids. Such mixed salts can be produced by cocrystallization of different keto acids.

[0062] According to the present invention, "branched chain keto acids" (BCKA) are understood to be keto analogs of BCAA, i.e., keto-leucine, keto-valine and keto-isoleucine and / or salts of these BCKAs. Salts of BCKAs should also include mixed salts of such BCKAs. Such mixed salts can be produced by co-crystallization of different BCKAs.

[0063] According to the present invention, "growth factor" should be understood as any naturally occurring substance capable of stimulating cell growth, proliferation and cell differentiation. Preferred growth factors are in the form of proteins or steroid hormones. According to one embodiment of the present invention, the expression "growth factor" should be interpreted as involving growth factors selected from the following: fibroblast growth factor (FGF), including acidic FGF and basic FGF, insulin, insulin-like growth factor (IGF), epithelial growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF) including TGFα and TGFβ, cytokines such as interleukin 1, 2, 6, granulocyte stimulating factor and leukocyte inhibitory factor (LIF).

[0064] The "sterile form" of a nutrient composition, culture medium, cell culture medium, etc. is understood to define the absence of any living organisms in said composition, culture medium, cell culture medium, etc.

[0065] In the context of the present invention, "solid medium" is understood to be any non-liquid or non-gas medium. Preferred solid medium of the present invention is a gel-like medium, such as agar-agar, carrageenan or gelatin.

[0066] In the context of the present invention, "passaging of cells" (also called subculturing or splitting of cells) is understood to mean the transfer of a small number of cells to a new culture vessel. If splitting is done regularly, cells can be cultured for longer periods of time, since it avoids senescence associated with prolonged high cell densities. Suspension cultures are easily passaged using small cultures containing a small number of cells diluted in a larger volume of fresh culture medium.

[0067] The present invention generally relates to a culture medium, preferably a cell culture medium, comprising at least one keto acid selected from the group consisting of ketoleucine, ketovaline, ketoisoleucine and ketophenylalanine and / or salts of these keto acids. The keto acid is preferably selected from the group consisting of the branched-chain keto acids ketoleucine, ketovaline and ketoisoleucine.

[0068] In a specific embodiment, the culture medium comprises a mixed salt of two or more keto acids. In this embodiment, two or more free keto acids selected from keto-leucine, keto-valine, keto-isoleucine and keto-phenylalanine are co-crystallized with one or more alkaline earth metal salts, preferably selected from calcium carbonate, calcium hydroxide, calcium acetate, calcium chloride, calcium oxide, magnesium hydroxide and magnesium acetate.

[0069] In a preferred configuration of the invention, the culture medium comprises ketoleucine, ketovaline and ketoisoleucine in an approximate ratio of about 2:1:1.

[0070] It is further preferred to use an alkali metal salt or alkaline earth metal salt of the keto acid. The preferred salt is Na + , K + , Ca 2+ and Mg 2+ Particularly preferred is the Na salt of the keto acid + or K + Salt.

[0071] In one embodiment, the culture medium further comprises at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffer and / or at least one vitamin. In a particularly preferred embodiment, the culture medium comprises all of at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffer and at least one vitamin.

[0072] According to another embodiment of the present invention, the culture medium is in liquid form, in the form of gel, powder, granules, pills or in the form of tablets.

[0073] In a preferred embodiment, the culture medium of the present invention is a defined culture medium or a serum-free culture medium. For example, the keto acids of the present invention can be supplemented to CHOMACS CD culture medium of Miltenyi Biotech (Bergisch Gladbach, Germany), PowerCHO-2 CD culture medium available from LONZA (Basel, Switzerland), Acti-CHO P culture medium of PAA (PAA Laboratories, Pasching, Austria), Ex-Cell CDCHO culture medium available from SAFC, SFM4CHO culture medium and CDM4CHO culture medium of ThermoFisher (Waltham, USA). The keto acids of the present invention can also be supplemented to DMEM culture medium (Life Technologies Corp., Carlsbad, USA). However, the present invention is not limited to the supplementation of the above culture medium.

[0074] In other preferred embodiments, the culture medium is a liquid culture medium in a 2-fold, 3-fold, 3.33-fold, 4-fold, 5-fold or 10-fold concentrated form (volume / volume) relative to the use concentration of the culture medium. This allows the preparation of a "ready-to-use" culture medium by simply diluting the concentrated culture medium with a corresponding volume of sterile water. Such a concentrated form of the culture medium of the present invention can also be used by adding it to a culture (e.g., in a batch feed culture process).

[0075] The culture medium of the present invention preferably contains all the nutrients required for sustained growth and product formation. The formulations for preparing culture medium, especially cell culture medium, are well known to those skilled in the art (see, for example, Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, Öztürk and Wei-Shou Hu eds., Taylor and Francis Group 2006). Various culture media can be commercially obtained from various sources.

[0076] The culture medium of the present invention preferably includes a carbohydrate source. The main carbohydrate used in the cell culture medium is glucose, which is routinely supplemented at 5 to 25 nM. In addition, any hexose, such as galactose, fructose or mannose or a combination can be used.

[0077] The culture medium also typically contains at least the essential amino acids (i.e., His, Ile, Leu, Lys, Met, Phe, Thr, Try, Val) as well as certain non-essential amino acids. Non-essential amino acids are often included in cell culture media if the cell line is not capable of synthesizing the amino acids, or if the cell line does not produce sufficient amounts of the amino acids to support maximal growth. Additionally, mammalian cells may also use glutamine as a primary energy source. Glutamine is typically included at higher concentrations than the other amino acids (2-8 mM). However, as mentioned above, glutamine can spontaneously break down to form ammonia, and some cell lines produce toxic ammonia more rapidly.

[0078] The culture medium of the present invention preferably contains salts. Salts are added to the cell culture medium to maintain isotonic conditions and prevent osmotic imbalance. The weight-gram molecular osmotic pressure concentration of the culture medium of the present invention is about 300mOsm / kg, although many cell lines can tolerate about 10% or more variation of this value. The weight-gram molecular osmotic pressure concentration of some insect cell cultures tends to be higher than 300 mOsm / kg, and this can be 0.5%, 1%, 2 to 5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30% higher than 300 mOsm / kg. The most commonly used salts in cell culture media include Na + , K + Mg 2+ , Ca 2+ , Cl - 、SO4 2- PO4 3- and HCO 3- (e.g. CaCl2, KCl, NaCl, NaHCO3, Na2HPO4).

[0079] Other inorganic elements may be present in the culture medium. They include Mn, Cu, Zn, Mo, Va, Se, Fe, Ca, Mg, Si and Ni. Many of these elements are involved in enzymatic activity. They may be provided in the form of salts, such as CaCl2, Fe(NO3)3, MgCl2, MgSO4, MnCl2, NaCl, NaHCO3, Na2HPO4 and trace elements such as ions of selenium, vanadium and zinc. These inorganic salts and trace elements may be commercially available, for example, from Sigma (Saint Louis, Missouri).

[0080] The culture medium of the present invention preferably contains vitamins. Vitamins are commonly used by cells as cofactors. Each cell line varies greatly in its vitamin requirements, although additional vitamins are generally required if the cell culture medium contains little or no serum, or if the cells are grown at high density. Exemplary vitamins that are preferably present in the culture medium of the present invention include biotin, choline chloride, folic acid, i-inositol, nicotinamide, D-Ca, 1,2-dihydrotestosterone ... ++ - Pantothenate, pyridoxal, riboflavin, thiamine, pyridoxine, niacinamide, A, B6, B12, C, D3, E, K and para-aminobenzoic acid (PABA).

[0081] The culture medium of the present invention may also contain serum. Serum is the supernatant of coagulated blood. Serum components include attachment factors, micronutrients (e.g., trace elements), growth factors (e.g., hormones, proteases), and protective elements (e.g., antitoxins, antioxidants, antiproteases). Serum can be obtained from a variety of animal sources, including human, bovine, or horse serum. When included in a cell culture medium according to the present invention, serum is typically added at a concentration of 5-10% (volume). Preferred cell culture media are serum-free.

[0082] To promote cell growth in the absence of serum or in a serum-reduced medium, one or more of the following polypeptides may be added to the cell culture medium of the present invention: for example, fibroblast growth factor (FGF) including acidic FGF and basic FGF, insulin, insulin-like growth factor (IGF), epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF) including TGFα and TGFβ, any cytokine such as interleukin 1, 2, 6, granulocyte stimulating factor, leukocyte inhibitory factor (LIF), etc.

[0083] In other embodiments, the culture medium does not contain polypeptides (ie, peptides having more than 20 amino acids).

[0084] The culture medium of the invention may also contain shorter chain peptides: examples include dipeptides of glutamine, tyrosine, cysteine, cysteine, asparagine and serine.

[0085] One or more lipids can also be added to the culture medium of the present invention, such as linoleic acid, linolenic acid, arachidonic acid, palmitoleic acid, oleic acid, polyenoic acid and / or fatty acids of 12, 14, 16, 18, 20 or 24 carbon atoms, each carbon atom is branched or unbranched), phospholipids, lecithin (phosphatidylcholine) and cholesterol. One or more of these lipids can be included in the serum-free medium as a supplement. Phosphatidic acid and lysophosphatidic acid stimulate the growth of certain anchor-dependent cells (such as MDCK, mouse epithelial and other kidney cell lines), while phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol stimulate the growth of human fibroblasts in serum-free medium. Ethanolamine and cholesterol are also shown to promote the growth of certain cell lines. In certain embodiments, the cell culture medium does not contain lipids.

[0086] One or more carrier proteins, such as bovine serum albumin (BSA) or transferrin, may also be added to the culture medium. Carrier proteins can aid in the transport of certain nutrients or trace elements. BSA is often used as a carrier for lipids that are insoluble in aqueous solution, such as linoleic acid and oleic acid. In addition, BSA can also be used as a carrier for certain metals, such as Fe, Cu, and Ni. In protein-free formulations, non-animal-derived BSA substitutes, such as cyclodextrin, can be used as lipid carriers.

[0087] One or more attachment proteins (eg, fibronectin, laminin, and pronectin) may also be added to the cell culture medium to help promote anchorage-dependent cell attachment to the substrate.

[0088] The culture medium may optionally contain one or more buffers. Suitable buffers include, but are not limited to, N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphates, bicarbonates, and other buffers suitable for cell culture applications. Suitable buffers are those that provide buffering capacity without substantial cytotoxicity to the cultured cells. The selection of suitable buffers is within the capabilities of those of ordinary skill in the art of cell culture.

[0089] Polyanionic or polycationic compounds can be added to the culture medium to prevent cell clumping and to promote the growth of cells in suspension.

[0090] In a preferred embodiment, the culture medium is in liquid form. However, the culture medium may also be a solid culture medium, such as a gelatinous culture medium, such as an agar-containing culture medium, a carrageenan-containing culture medium or a gelatin-containing culture medium.

[0091] Preferably, the culture medium is in sterile form.

[0092] In a preferred embodiment of the present invention, the keto acid is present in the culture medium in a concentration of 0.01 to 10 g / l, or 0.1 to 5 g / l, or 0.1 to 0.5 g / l, preferably 0.01 to 0.2 g / l.

[0093] The above concentrations are given as concentrations in non-concentrated medium (i.e. concentrations present in actual culture). Concentrated medium may contain X times higher concentrations.

[0094] In an alternative embodiment of the present invention, keto acids are supplemented into the culture medium containing the non-keto analogs of deficiency, respectively. In a specific embodiment, the culture medium comprises both ketoleucine and leucine and / or both ketovaline and valine and / or both ketoisoleucine and isoleucine and / or both ketophenylalanine and phenylalanine. In another embodiment, the amino acid is replaced by the keto analogs in the culture medium. In a specific embodiment, the culture medium comprises ketoleucine instead of leucine and / or comprises ketovaline instead of valine and / or comprises ketoisoleucine instead of isoleucine and / or comprises ketophenylalanine instead of phenylalanine.

[0095] Substratum of the present invention can be concentrated form.It can be for example 2 times, 3 times, 3.33 times, 4 times, 5 times, 10 times, 20 times, 50 times or 100 times concentrated form (relative to the concentration of supporting the growth of cells and product formation).Such concentrated medium helps to prepare the substratum for use by diluting the concentrated medium with aqueous solvents such as water.Such concentrated medium can be used for batch culture, but can also be advantageously used for batch feeding or continuous culture, wherein concentrated nutrients are added to the ongoing cell culture, for example to supplement the nutrients consumed by the cells during the culture period.

[0096] In other embodiments of the present invention, the culture medium is in a dry form, such as in a dry powder form, or in a granule form, or in a pellet form, or in a tablet form.

[0097] The present invention also relates to the use of the culture medium of the present invention for culturing cells. Another aspect of the present invention relates to the use of the culture medium of the present invention for producing a cell culture product.

[0098] A preferred embodiment of the present invention relates to the use of the culture medium according to the present invention for culturing animal cells or plant cells, most preferably mammalian cells. In a specific embodiment, the cells to be cultured are CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, insect cells, fibroblasts, muscle cells, nerve cells, stem cells, skin cells, endothelial cells and hybridoma cells. Preferred cells of the present invention are CHO cells and HEK cells.

[0099] Also included within the scope of the present invention is a method of culturing cells, the method comprising contacting the cells with a cell culture medium according to the present invention. In one embodiment of the present invention, the method of culturing cells comprises contacting the cells with a basal culture medium under conditions that support cell culture, and supplementing the basal cell culture medium with a concentrated culture medium according to the present invention. In a preferred embodiment, the basal culture medium is supplemented with a concentrated feed or culture medium for more than one day.

[0100] Another aspect relates to a method for producing a culture medium according to the invention, wherein the culture medium comprises at least one keto acid according to the invention. The method for producing a culture medium according to the invention comprises at least one step of adding at least one keto acid according to the invention to the culture medium. Similarly, one aspect of the invention relates to the use of a keto acid according to the invention for producing a cell culture medium.

[0101] Another aspect relates to a method of modifying a culture medium, wherein said modification of said culture medium comprises adding to said culture medium at least one keto acid of the present invention.

[0102] Another aspect relates to a method for producing a liquid culture medium, the method comprising providing a solid culture medium according to the present invention (e.g. in the form of a dry powder, or in the form of granules, or in the form of pills or in the form of tablets); and dissolving the solid culture medium in an aqueous medium, such as water.

[0103] Another aspect of the present invention relates to the use of a keto acid according to the present invention in a culture medium for culturing cells. Another aspect of the present invention relates to the use of a keto acid according to the present invention for cell culture.

[0104] The present invention also relates to a method for producing a cell culture product, comprising the following steps: (i) providing cells capable of producing the cell culture product; (ii) contacting the cells with a culture medium of the present invention; and (iii) obtaining the cell culture product from the culture medium or from the cells. Similarly, the present invention relates to the use of a keto acid according to the present invention for preparing a cell culture product.

[0105] In preferred methods, the cell culture product is a therapeutic protein, a diagnostic protein, a polysaccharide such as heparin, an antibody, a monoclonal antibody, a growth factor, an interleukin, a virus, a virus-like particle, or an enzyme.

[0106] The cell culture according to the present invention can be carried out as a batch culture, a fed-batch culture or a continuous culture. Example

[0107] Example 1: Production of AAV9 and AAV2 in Human Embryonic Kidney (HEK) Cells Viral vectors (AAV2)

[0108] Human embryonic kidney (HEK 293T) cells were cultured in serum-free medium (FreeStyle 293 Expression Medium, Thermo Fisher Scientific) with or without the addition of 1 mM BCKA mixture in the form of potassium salts. The mixture used contained K of the branched-chain keto acids keto-leucine:keto-isoleucine:keto-valine in a ratio of 2:1:1. + Cells were transfected with plasmid DNA to produce viral vectors of adeno-associated virus (AVV) type 9 (AAV9) and adeno-associated virus type 2 (AAV2) in the presence or absence of a branched-chain keto acid mixture. The amount of plasmid produced in HEK 293T cells was determined using quantitative real-time polymerase chain reaction (qPCR).

[0109] For qPCR analysis, cultured cells were scraped from the wells and pelleted by centrifugation at 1,500 rpm for 5 minutes. The supernatant was removed until 1.0 ml of medium remained; the cells were resuspended and frozen at -80°C. To ensure lysis of the cells, they were frozen and thawed three times. After the final thaw, the cells were centrifuged at 1,000 × g for 5 minutes, and the supernatant was transferred to a new tube. To remove any unencapsulated free viral DNA and capsids from intact viral particles, the supernatant was treated with DNase and proteinase K. Real-time qPCR was performed to detect the poly A region of AAV DNA.

[0110] Figure 1 Viral vector titers of adeno-associated virus (AVV) type 9 (AAV9) and adeno-associated virus type 2 (AAV2) in the absence and presence of a mixture of potassium salts of branched-chain keto acids (BCKAs) are shown. Viral vector titers measured in serum-free medium without added BCKAs were normalized to 1 to show the change in viral vector titer compared to the addition of BCKAs to the medium. Unexpectedly, the addition of a mixture of potassium salts of BCKAs to the serum-free medium used to culture the cells resulted in 2.3-fold higher viral vector titers for AAV2 and 3.7-fold higher viral vector titers for AAV9.

[0111] Example 2: Infection with AAV9 and AAV2 Virus Vectors Human embryonic kidney (HEK) cells

[0112] Viral particles produced by transfected HEK 293T cells as described in Example 1 were harvested and fresh HEK293T cells were incubated with viral supernatants in serum-free medium in the absence or presence of the BCKA potassium salt mixture also used in Example 1. The infectivity of the viral particles was analyzed using a luciferase assay. The luciferase gene was also incorporated into the viral vector, and the luciferase activity detected was proportional to the amount of luciferase produced and was used as a readout for the uptake of viral particles in the cells.

[0113] Figure 2 Viral vector infectivity (measured by luciferase activity) of adeno-associated virus type 9 (AVV) (AAV9) and adeno-associated virus type 2 (AAV2) in the absence and presence of a BCKA potassium salt mixture is shown. The infectivity determined in serum-free medium without the addition of BCKA was normalized to 1 to show the change in viral vector infectivity compared to the addition of BCKA to the medium. Unexpectedly, the addition of a BCKA potassium salt mixture to the serum-free medium used to culture cells resulted in a 1.5-fold increase in infectivity for AAV2 and a 1.65-fold increase in infectivity for AAV9.

[0114] Example 3: Antibody production in Chinese hamster ovary (CHO) cells in the presence of different concentrations of ketoleucine

[0115] Antibody-producing Chinese hamster ovary (CHO) cells (DG44 subclone) were cultured in shake flasks (125 ml or 250 ml with 50 ml or 100 ml culture volume, respectively) in TC42 / CHOMACS CD medium (TeutoCell AG) containing 8 mM L-glutamine. The medium was prepared by mixing two solutions. The first contained a conventional medium preparation (containing L-leucine and calcium chloride). In the second, the amino acid L-leucine in the medium was replaced with an equimolar amount of keto-leucine (178 mg / l) as a calcium salt. These solutions were mixed in different ratios. The growth of cells cultured in the different mixtures was compared with cells cultured in conventional medium.

[0116] Figure 3 The overall viable cell density of CHO cells cultured in medium containing different amounts of keto-leucine instead of L-leucine is shown. The X-axis shows 10 per ml. 6 The total viable cell density (VCD) of cells was calculated using the table 1. A corresponds to cells cultured in a medium containing 178 mg / L ketoleucine, B corresponds to a mixture containing a 30% (v / v) ketoleucine solution, C corresponds to a mixture containing a 5% (v / v) ketoleucine solution and the reference contains only L-leucine. The final concentration of ketoleucine in the final medium is shown in Table 1. The viable cell density was slightly reduced for the medium containing ketoleucine instead of L-leucine. However, it can be clearly demonstrated that the amino acid L-leucine can be replaced by the keto analogue ketoleucine.

[0117] Table 1: Keto-leucine (keto-leu) concentration in the final culture medium

[0118] condition Final Keto-Leu Concentration A 178 mg / L B 53.4 mg / L C 8.9 mg / L Reference 0 mg / L

[0119] Figure 4The results show that CHO cells cultured in medium containing different amounts of keto-leucine instead of L-leucine (eg Figure 3 The relative concentration of antibody produced by cells cultured in a reference medium containing L-leucine (reference) was normalized to 1.0. This clearly shows that L-leucine can be replaced by the branched-chain keto acid ketoleucine in the cultured CHO cells used for antibody production.

[0120] Example 4: Antibody production in Chinese hamster ovary (CHO) cells in the presence of different concentrations of ketovaline

[0121] Antibody-producing Chinese hamster ovary (CHO) cells (DG44 subclone) were cultured in shake flasks (125 ml or 250 ml with 50 ml or 100 ml culture volume, respectively) in TC42 / CHOMACS CD medium (TeutoCell AG) containing 8 mM L-glutamine. The medium was prepared by mixing two solutions. The first contained a conventional medium formulation (containing L-valine and calcium chloride). In the second, 163 mg / L of keto-val was added as a calcium salt to the conventional medium. These solutions were mixed in different ratios. Figure 5 The maximum viable cell density of CHO cells cultured in medium containing different amounts of ketovaline in addition to L-valine is shown. The X-axis shows 10 cells per milliliter. 6 The maximum viable cell density (VCD) of cells was calculated. A represents a solution containing L-valine and 163 mg / L of ketovaline. B is a mixture containing 70% (v / v) of medium with ketovaline and 30% (v / v) of regular medium. C is a mixture of 55% (v / v) of medium with ketovaline and 45% (v / v) of regular medium. D contains a 5% (v / v) ketovaline solution, while the reference corresponds to regular medium as described above. The final concentration of ketovaline in the final medium is shown in Table 2.

[0122] Table 2: Keto-Val concentration in final culture medium

[0123] Final keto-val concentration A 163 mg / L B 114.1 mg / L C 89.65 mg / L D 8.15 mg / L Reference 0 mg / L

[0124] Figure 6 The results show that CHO cells cultured in medium containing different amounts of keto-valine and L-valine (eg Figure 5 The relative concentration of antibodies produced by cells cultured in the reference was normalized to 1.0. It can be clearly shown that antibody production is enhanced in the presence of ketovaline compared to cells cultured in the absence of ketovaline.

[0125] Example 5: Effect of BCKA on cytokine secretion

[0126] First, macrophages were obtained from THP-1 monocytes by differentiation. When studying the preventive regimen, Ca2+ containing branched-chain ketoacids keto-leucine:keto-isoleucine:keto-valine (BCKA) in a ratio of 2:1:1 was added 24 h before induction of immune response by LPS. + For the treatment with BCKA, different time periods were selected before and continuously. When LPS treatment was started, the treatment with BCKA was stopped (BCKA treatment before LPS stimulation) or the treatment with BCKA was continued (BCKA treatment continued to LPS stimulation). To evaluate the level of immune response, the proinflammatory cytokines TNF-α, IL-1β and IL-6 were measured in the cell culture supernatant. The expression of proinflammatory cytokines in cells treated with BCKA was as shown in Figure 2. Figure 1 shown.

[0127] Figure 7 The effect of 100 µg / ml BCKA on the secretion of three proinflammatory cytokines is shown. The amount of cytokines was measured in three independent assays, each in triplicate, and normalized to the number of cells in each corresponding sample. Error bars represent standard deviation.

[0128] Positive and negative controls were not exposed to BCKA at any time and showed LPS-triggered cytokine release relative to the stimulated baseline. Cells treated with BCKA and then treated with LPS as an inflammatory stimulus were marked "pre-inflammatory (+)", and cells not exposed to LPS but also treated with BCKA were marked "pre-inflammatory (-)". The latter served as a control to examine whether BCKA itself caused cytokine release or interfered with the assay in another way. This was excluded because cells treated with natural compounds showed the same cytokine profile as the negative control. In the case of pretreatment with BCKA before LPS stimulation, a significant reduction in cytokine release can be observed. This confirms the significant effect of previous and continuous treatment with BCKA on the immune response. In addition, similar results were obtained using the single keto acids ketovaline, ketoleucine and ketoisoleucine.

[0129] The expression of pro-inflammatory cytokines in cells treated with keto-valine is shown in Figure 8 . Fig. 9 The expression of pro-inflammatory cytokines in cells treated with keto-leucine is shown. Fig.10 In the experiment, cells were treated with ketoisoleucine.

[0130] Figure 8The effect of 100 µg / ml of keto-valine on the secretion of three pro-inflammatory cytokines is shown. The amount of cytokines was measured in three independent assays, each in duplicate, and normalized to the number of cells in each corresponding sample. Error bars represent standard deviation.

[0131] Fig. 9 The effect of 100 µg / ml of keto-leucine on the secretion of three pro-inflammatory cytokines is shown. The amount of cytokines was measured in three independent assays, each performed in duplicate, and normalized to the number of cells in each corresponding sample.

[0132] Fig.10 The effect of 100 µg / ml of ketoisoleucine on the secretion of three proinflammatory cytokines is shown. The amount of cytokines was measured in three independent assays, each in duplicate, and normalized to the number of cells in each corresponding sample. Error bars represent standard deviation.

Claims

1. A culture medium, such as a cell culture medium, comprising at least one keto acid selected from the group consisting of ketoleucine, ketovaline, ketoisoleucine and ketophenylalanine and / or salts of these keto acids.

2. The culture medium of claim 1, wherein the keto acid is a branched chain keto acid selected from the group consisting of ketoleucine, ketovaline, and ketoisoleucine.

3. The culture medium according to any one of the preceding claims, wherein the culture medium comprises ketoleucine, ketovaline and ketoisoleucine in an approximate ratio of 2:1:

1.

4. The culture medium according to any one of the preceding claims, comprising an alkali metal salt or alkaline earth metal salt of the keto acid.

5. The culture medium according to any one of the preceding claims, comprising Na + , K + , Ca 2+ and Mg 2+ Salt, preferably Na + or K + Salt.

6. The culture medium according to any of the preceding claims, further comprising at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffer and / or at least one vitamin.

7. The culture medium according to any one of the preceding claims, wherein the culture medium is in the form of a liquid, a gel, a powder, a granule, a pill or a tablet.

8. The culture medium according to any one of the preceding claims, wherein the culture medium is a serum-free medium or a defined medium.

9. The culture medium according to any of the preceding claims, wherein the keto acid is present in the culture medium in a concentration of 0.01 to 10 g / l, or 0.1 to 5 g / l, or 0.1 to 0.5 g / l, preferably 0.01 to 0.2 g / l.

10. The culture medium of any one of the preceding claims, wherein the culture medium is in a 2-fold, 3-fold, 3.33-fold, 4-fold, 5-fold or 10-fold concentrated form relative to the use concentration of the culture medium.

11. Use of the culture medium according to any one of claims 1 to 9 for culturing cells.

12. The use according to claim 11, wherein the cell is an animal cell or a plant cell, preferably a mammalian cell.

13. The use of claim 12, wherein the cell is selected from the group consisting of: CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, insect cells, fibroblasts, muscle cells, neural cells, stem cells, skin cells, endothelial cells and hybridoma cells.

14. Use of at least one keto acid selected from the group consisting of ketoleucine, ketovaline, ketoisoleucine and ketophenylalanine and / or salts of these keto acids in a culture medium for culturing cells.

15. A method for producing a cell culture product, comprising the steps of: providing cells capable of producing the cell culture product; contacting the cells with the culture medium of any one of claims 1 to 9; and The cell culture product is obtained from the culture medium or from the cells.

16. The method of claim 15, wherein the cell culture product is selected from the group consisting of therapeutic proteins, diagnostic proteins, polysaccharides such as heparin, antibodies, monoclonal antibodies, growth factors, interleukins, viruses, virus-like particles and enzymes.

Images