Cell culture media comprising keto acids

Abstract

To provide cell culture media comprising alpha keto acids.SOLUTION: The poor solubility of some amino acids like isoleucine, leucine and valine can be overcome by substituting them with the respective alpha keto acids.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cell culture media containing alpha keto acids. The poor solubility of some amino acids, such as isoleucine, leucine, and valine, can be overcome by replacing them with the respective alpha keto acids. [Background technology]

[0002] Cell culture media supports and maintains the growth of cells in an artificial environment. Depending on the type of organism whose growth is being supported, cell culture media contain a complex mixture of components, sometimes over 100 different components. The cell culture media required for the growth of mammalian, insect, or plant cells are typically much more complex than media to support bacterial and yeast growth.

[0003] The first cell culture media developed consisted of undefined components such as plasma, serum, embryonic extracts, or undefined biological extracts or peptones. Thus, significant progress has been made with the development of chemically defined media, which often contain, but are not limited to, amino acids, vitamins, metal salts, antioxidants, chelating agents, growth factors, buffers, hormones, and many other substances known to those skilled in the art.

[0004] Some cell culture media are proposed as sterile aqueous liquids. The disadvantages of liquid cell culture media are their short shelf life and the difficulty of transporting and storing them. As a result, many cell culture media are currently proposed as finely ground dry powder mixtures. These are manufactured for dissolution in water and / or aqueous solutions, and in the dissolved state, are often designed, together with other supplements, to replenish cells with a substantial nutrient base for the growth and / or production of biopharmaceuticals from said cells.

[0005] Many biopharmaceutical production platforms are based on fed-batch cell culture protocols. The goal is typically to develop high-titer cell culture processes that meet growing market demands and reduce manufacturing costs. Achieving maximum production capacity requires the use of high-performance recombinant cell lines as well as improvements in cell culture media and process parameters.

[0006] In a fed-batch process, a basal medium supports initial growth and production, and a feed medium prevents nutrient starvation and sustains the production phase. Media are selected to accommodate distinct metabolic requirements during the various production phases. The setting of process parameters—including feeding strategies and control parameters—defines the chemical and physical environment favorable for cell growth and protein production.

[0007] Optimization of the feed medium is a major aspect in the optimization of a fed-batch process. In most cases, the feed medium is highly concentrated to avoid dilution of the recombinant protein in the bioreactor. The controlled addition of nutrients directly affects the growth rate, viability and titer of the culture.

[0008] However, other cell culture processes, such as batch and perfusion processes, also require precisely composed and often highly concentrated media formulations. Particularly in perfusion processes, regular media exchange in bioreactors requires operators to prepare and handle large volumes of liquid media. To reduce the footprint required to store these volumes, media concentration is required.

[0009] A limiting factor for the preparation of cell culture media from dry powders is the poor solubility or stability of some components, especially some amino acids. Consequently, it is desirable to find a method that provides a dry powder medium composition that is sufficiently soluble to produce a highly concentrated liquid medium composition. It has been found that the amino acids isoleucine, leucine, valine, phenylalanine and methionine can be substituted with their respective alpha keto acids without any negative effects and in some cases even with positive effects on cell growth and improved solubility. Moreover, these keto acids have even been found to have a stabilizing effect on liquid cell culture media formulations.

[0010] In 1959, a paper dealing with amino acid metabolism described the possibility of substituting some amino acids with their keto acids (Eagle H: Amino acid metabolism in mammalian cell cultures. Science 1959, 130(3373):432-437). However, since then, no attention has been paid to the possibility that certain keto acids can be used as amino acid substitutes in high-performance cell cultures and that they are suitable for overcoming the solubility and stability problems of some amino acids. Summary of the Invention

[0011] The present invention is therefore directed to a dry powder or dry granule cell culture medium comprising at least one alpha keto acid from the group of 4-methyl-2-oxopentanoic acid (ketoLeu), 3-methyl-2-oxopentanoic acid (ketoIle), alpha-ketoisovaleric acid (ketoVal), phenylpyruvic acid (ketoPhe) and alpha-ketogamma-methylthiobutyric acid (ketoMet), and / or derivatives thereof, in an amount such that the concentration of each keto acid and / or derivative thereof in the liquid medium obtained after dissolution of the dry powder or dry granule cell culture medium is greater than 10 mM, preferably 20-600 mM, most preferably 30-300 mM. Typically, each keto acid is present at a different concentration, where typically 4-methyl-2-oxopentanoic acid (ketoLeu), 3-methyl-2-oxopentanoic acid (ketoIle), alpha-ketoisovaleric acid (ketoVal), phenylpyruvic acid (ketoPhe) and / or their derivatives are present at higher concentrations of greater than 50 mM, and where alpha-ketogamma-methylthiobutyric acid (ketoMet) is typically present at a lower concentration, typically 10-30 mM.

[0012] In a preferred embodiment, when the dry powder or dry granule cell culture medium is a feed medium, it contains less than 30 mol % of the corresponding amino acid compared to the keto acid and / or derivative, which means that the molar ratio of the two compounds is less than 3:10. In another embodiment, the dry powder or dry granule cell culture feed medium does not contain the corresponding amino acid. In other media, such as perfusion media or (fed-)batch basal media, it may be preferable to have both amino acids and the corresponding keto acids and / or their derivatives in the media formulation.

[0013] In another embodiment, the dry powder or dry granule cell culture medium comprises two or more alpha keto acids and / or their derivatives. In a preferred embodiment, the dry powder or dry granular cell culture medium comprises the sodium salt of one or more of the above-listed alpha keto acids. In a preferred embodiment, the dry powder or dry granule cell culture medium comprises one or more alpha keto acids selected from 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid and / or salts thereof, preferably sodium salts thereof.

[0014] The present invention is directed to a method for stabilizing a liquid cell culture medium comprising at least 20 mM, preferably 30-600 mM, of one or more alpha-keto acids selected from 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid and / or their derivatives, preferably 4-methyl-2-oxopentanoic acid and / or 3-methyl-2-oxopentanoic acid and / or alpha-ketoisovaleric acid and / or their derivatives, wherein the resulting medium exhibits less color change and / or less precipitation when stored at 4° C. or at room temperature for 90 days compared to a medium of otherwise identical composition lacking the keto acids and / or their derivatives or in which the keto acids and / or their derivatives are substituted with the corresponding amino acids and / or their derivatives.

[0015] The present invention is further directed to a method for improving the solubility of a dry powder or dry granule cell culture medium of defined composition by completely or partially replacing one or more of the amino acids isoleucine, leucine, valine, phenylalanine and methionine with the corresponding keto acids selected from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid and / or their derivatives.

[0016] In a preferred embodiment, at least 50%, more preferably 70%, and most preferably at least 90% (molar ratio) of each amino acid is substituted with the corresponding alpha keto acid and / or a derivative thereof. In this case, substitution means that at least 80 mol%, typically approximately 100 mol%, of the corresponding keto acid and / or its derivative is added to the medium in place of a given amount of amino acid, and preferably 100 to 150 mol% of the corresponding keto acid and / or its derivative is added to the medium.

[0017] In a preferred embodiment, the method comprises providing a dry powder or dry granule cell culture medium in which amino acids have been substituted as described above, and dissolving said medium, wherein dissolution occurs faster and / or with less liquid than a medium of otherwise identical composition in which the amino acids have not been substituted. In another preferred embodiment, the dry powder or dry granular medium is dissolved to provide a liquid medium having a pH of 8.5 or less. In a preferred embodiment, it dissolves to give a liquid medium having a pH of 6.5 to 8.5, most preferably 6.7 to 7.8.

[0018] In one embodiment, the dry powder or dry granule cell culture medium with improved solubility includes 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.

[0019] In another embodiment, the solubility-improved dry powder or dry granule cell culture medium dissolves to provide a liquid medium containing solid components that are soluble in the solvent at 50-400 g / l, preferably 100-300 g / l, and / or the concentration of each keto acid and / or its salt is greater than 10 mM, preferably 30-600 mM.

[0020] The present invention further comprises: a) mixing at least one alpha-keto acid from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid and / or derivatives thereof with other components of the cell culture medium; b) subjecting the mixture of step a) to grinding. The present invention is directed to a method for producing a dry powder cell culture medium according to the present invention,

[0021] In a preferred embodiment, step b) is carried out in a pin mill, a Fitz mill or a jet mill. In another preferred embodiment, the mixture from step a) is cooled to a temperature below 0° C. prior to grinding.

[0022] The present invention provides a) providing a bioreactor; b) mixing the cells to be cultured with a liquid cell culture medium in which one or more of the amino acids isoleucine, leucine, valine, phenylalanine and methionine have been partially or totally replaced by the corresponding ketoacids selected from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid, and / or derivatives thereof; c) incubating the mixture of step b). The present invention is further directed to a process for culturing cells according to the present invention.

[0023] In preferred embodiments, the liquid cell culture medium comprises each keto acid and / or its derivatives present at a concentration greater than 10 mM. The present invention also provides - loading the cells and aqueous cell culture medium into the bioreactor - Incubating the cells in a bioreactor - adding cell culture medium, in this case a feed medium, to the bioreactor continuously throughout the incubation of the cells in the bioreactor or once or several times during said incubation period wherein the feed medium has a pH of less than pH 8.5 and comprises at least one alpha-keto acid from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid and / or derivatives thereof, The present invention is also directed to a fed-batch process for culturing cells in a bioreactor, according to

[0024] Preferably, the feed medium contains at least 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid and / or salts thereof, each at a concentration of 20 to 600 mmol / L, preferably 20 to 400 mmol / L. The present invention is further directed to a perfusion process using a liquid cell culture medium in which one or more of the amino acids isoleucine, leucine, valine, phenylalanine, and methionine are partially or totally replaced by the corresponding keto acid selected from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid, and alpha-keto gamma methylthiobutyric acid, and / or derivatives thereof. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 shows the determination of the maximum solubility of Ile or ketoIle in the Ile- and Leu-deficient Cellvento® 4Feed formulation (125 g / L, pH 7.0 + / - 0.2). Solutions with a turbidity of less than 5 NTU are considered soluble. [Figure 2] Figure 2 shows the determination of maximum solubility of Leu or keto-Leu in a Cellvento® 4Feed formulation (125 g / L, pH 7.0 + / - 0.2) depleted of Ile and Leu. Solutions with a turbidity of less than 5 NTU are considered soluble. Further information on Figures 1 and 2 can be found in Example 2.

[0026] [Figure 3] Figure 3 shows the solubility limit of Cellvento® 4 Feed at pH 7.0. Turbidity was measured using a turbidity meter. Further details can be found in Example 3. [Figure 4] Figure 4 shows the solubility limit at pH 7.0 of a modified 4Feed formulation in which Ile and Leu are replaced by ketoIle and ketoLeu. Turbidity was measured using a turbidimeter. Further details can be found in Example 3.

[0027] [Figure 5] Figure 5A shows the baseline-corrected area under the curve for absorbance from 300 to 600 nm over time (DO-D90) AUC for a control feed containing Leu and a test feed in which Leu was depleted and replaced with an equimolar concentration of keto-Leu. Figure 5B shows the baseline-corrected area under the curve for absorbance from 300 to 600 nm over time (DO-D90) AUC for a control feed containing isoleucine and a test feed in which Leu was depleted and replaced with an equimolar concentration of keto-Ieu. Details can be found in Example 4.

[0028] [Figure 6] Figure 6A shows the area under the curve (D0-D90) of NH3 concentration measured in a feed containing keto-Leu compared to a control. The feed was stored for 3 months at 4°C and RT, and either protected from light or exposed to light. Figure 6B shows the area under the curve (D0-D90) of NH3 concentration measured in a feed containing keto-Ile compared to a control. The feed was stored for 3 months at 4°C and RT, and either protected from light or exposed to light. Details can be found in Example 4.

[0029] [Figure 7]Figure 7A: VCD over a 17-day fed-batch process in which Leu and Ile in the feed were replaced with either keto-Leu or keto-Ile, respectively. Starved 4Feed is a negative control and does not contain any Leu or Ile. Figure 7B: IgG produced over a 17-day fed-batch process in which Leu and Ile in the feed were replaced with either keto-Leu or keto-Ile, respectively. Details can be found in Example 5.

[0030] [Figure 8] Figure 8: Average specific productivity of a 17-day fed-batch process where Leu and Ile in the feed were replaced with either keto-Leu or keto-Ile, respectively. [Figure 9] Figure 9A: NH3 production during a 17-day fed-batch process in which Leu and Ile in the feed were replaced with either keto-Leu or keto-Ile, respectively. Figure 9B: Leu quantification in spent medium during a 17-day fed-batch process in which Leu and Ile in the feed were replaced with either keto-Leu or keto-Ile, respectively. [Figure 10] Figure 10A: Ile quantification in spent medium during a 17-day fed-batch process in which Leu and Ile in the feed were replaced with either keto-Leu or keto-Ile, respectively. Figure 10B: Allo-Ile quantification in spent medium during a 17-day fed-batch process in which Ile in the feed was replaced with keto-Ile.

[0031] [Figure 11] Figure 11: Glycosylation of IgG1 produced in a control process or in a process using a feed deficient in Ile / Leu and supplemented with either keto-Leu or keto-Ile. Glycoform distribution was determined using APTS labeling and CGE-LIF detection.

[0032] [Figure 12]Figure 12A: Aggregation and fragmentation of IgG1 produced by a control process or a process using a feed deficient in Ile / Leu and supplemented with keto-Leu or keto-Ile. High molecular weight (HMW) and low molecular weight (LMW) were determined using size exclusion chromatography. Figure 12B: Charge variants of IgG1 produced by a control process or a process using a feed deficient in Ile / Leu and supplemented with either keto-Leu or keto-Ile. Charge variant distribution was determined using cIEF on a capillary electrophoresis CESI 8000. Further details can be found in Example 5.

[0033] [Figure 13] Figure 13: Performance of keto-Leu-containing processes in the IgG1-expressing CHODG44 cell line compared to the control. [Figure 14] Figure 14: Performance of keto-Leu containing processes in the CHOK1 non-GS cell line expressing IgG1 compared to the control. Further details can be found in Example 6.

[0034] [Figure 15] Figure 15A: Batch experiment using CHOK1GS cell line cultured in medium containing Leu and Ile (control), or medium in which Ile or Leu was replaced by their equimolar concentrations of keto-Ile or keto-Leu. The seeding density was 0.2 million cells / mL, and VCD was measured using a Vi-CELL XR. Figure 15B: IgG concentration measured during the batch experiment. IgG was measured using a turbidity assay on a Cedex Bio HT (Roche). Further details can be found in Example 7.

[0035] [Figure 16] Figure 16: Batch experiments at higher seeding densities and various Leu / ketoLeu ratios. VCD and titer were measured, as well as released leucine in spent medium. Further details can be found in Example 7. [Figure 17]Figure 17: Replacement of Val with ketoVal in the feed. VCD and titer were measured, as well as released Val and NH concentrations in spent medium. Further details can be found in Example 8.

[0036] [Figure 18] Figure 18: Replacement of Phe in the feed with phenylpyruvate at either the same molar concentration as Phe (1x) or twice the concentration (2x). VCD and titer were measured, as well as released Phe concentration in spent medium. Further details can be found in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0037] A cell culture medium according to the present invention is any mixture of components that maintain and / or support the in vitro growth of cells. It can be a complex medium or a chemically defined medium. A cell culture medium can contain all components necessary to maintain and / or support the in vitro growth of cells, or it can contain only some components so that additional components can be added separately. Examples of cell culture media according to the present invention are complete media containing all components necessary to maintain and / or support the in vitro growth of cells, as well as medium supplements or feeds. In preferred embodiments, the cell culture medium is a complete medium, a perfusion medium, or a feed medium. Complete media, also called basal media, typically have a pH of 6.7 to 7.8. Feed media preferably have a pH below 8.5.

[0038] Typically, the cell culture medium according to the present invention is used to maintain and / or support the growth of cells in a bioreactor. A feed or feed medium is not a basal medium that supports initial growth and production in a cell culture, but rather a cell culture medium that is added at a later stage to prevent nutrient depletion and maintain the production phase, but rather a medium that is added at a later stage to sustain the production phase. A feed medium may have a higher concentration of some components than a basal culture medium. For example, some components, such as nutrients including amino acids or carbohydrates, may be present in a feed medium at about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or about 1000X the concentration of the basal medium.

[0039] Mammalian cell culture media is a mixture of components that maintain and / or support the growth of mammalian cells in vitro. Examples of mammalian cells are human or animal cells, preferably CHO cells, COS cells, IVERO cells, BHK cells, AK-1 cells, SP2 / 0 cells, L5.1 cells, hybridoma cells or human cells.

[0040] 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 known. Chemically defined media do not contain any yeast, animal, or plant tissue; they do not contain feeder cells, serum, hydrolysates, extracts, or digests, or other incompletely defined components. Chemically undefined or incompletely defined chemical components are those whose chemical composition and structure are unknown, exist in varying compositions, or can only be defined through extensive experimental effort comparable to evaluating the chemical composition and structure of proteins such as insulin, albumin, or casein.

[0041] Powdered cell culture media, or dry powder media, are cell culture media that typically result from a grinding or freeze-drying process. This means that powdered cell culture media are granular, particulate media—not liquid media. The term "dry powder" may be used interchangeably with the term "powder"; as used herein, "dry powder" refers simply to the overall appearance of the granular material and is not intended to imply that the material is completely free of complexed or aggregated solvents, unless otherwise indicated.

[0042] Dry granular medium is a dry medium resulting from a wet or dry granulation process. Preferably, it is a medium resulting from roller compaction of a dry powder medium. As used herein, the term dry simply refers to the overall appearance of the granular material and is not intended to mean that the material is completely free of complexed or aggregated solvents, unless otherwise indicated.

[0043] The cells cultured using the media according to the invention may be prokaryotic cells, such as bacterial cells, or eukaryotic cells, such as plant or animal cells. The cells may be normal, immortalized, abnormal, transformed, mutated, somatic, germ, stem, progenitor, or embryonic cells, any of which may be an established or transformed cell line, or may be obtained from a natural source.

[0044] Particle size refers to the average diameter of the particles, which is determined by laser light scattering (Mastersizer 3000, Malvern). The color change of the liquid cell culture medium is preferably determined visually or spectroscopically. Precipitation can be determined visually or by turbidimetry.

[0045] The inert atmosphere is created by filling each container or apparatus with an inert gas. Suitable inert gases are noble gases such as argon or, preferably, nitrogen. These inert gases are non-reactive and prevent undesired chemical reactions from occurring. In the process according to the present invention, the creation of an inert atmosphere means that the oxygen concentration is reduced to an absolute value of less than 10% (v / v), for example, by introducing liquid nitrogen or nitrogen gas.

[0046] Various types of mills are known to those skilled in the art. Pin mills, also known as centrifugal impact mills, use protruding pins on a rapidly rotating disk to provide the breaking energy that pulverizes solids. For example, pin mills are sold by Munson Machinery (USA), Premium Pulman (India), or Sturtevant (USA). Jet mills utilize compressed gas to accelerate particles and cause them to collide with each other in a process chamber. Jet mills are sold, for example, by Sturtevant (USA) or PMT (Austria). The Fitzmill, marketed by Fitzpatrick (USA), uses a rotor with blades to grind.

[0047] A continuously run process is one that is not run batchwise. When a milling process is run continuously, it means that the media components are constantly and steadily fed to the mill over a period of time. Cell culture media according to the invention, particularly complete media, typically include 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.

[0048] The medium may also contain surface-active components such as sodium pyruvate, insulin, vegetable proteins, fatty acids and / or fatty acid derivatives and / or pluronic acid and / or chemically prepared non-ionic surfactants. One example of a suitable non-ionic surfactant is a difunctional block copolymer surfactant terminated in primary hydroxyl groups, also known as poloxamer (e.g., available from BASF, Germany under the trade name pluronic®).

[0049] The sugar components are all monosaccharides or disaccharides, such as glucose, galactose, ribose, or fructose (examples of monosaccharides), or sucrose, lactose, or maltose (examples of disaccharides). Examples of amino acids according to the invention are tyrosine, protein organic amino acids, in particular the essential amino acids leucine, isoleucine, lysine, methionine, phenylalanine, arginine, threonine, tryptophan, and valine, as well as non-protein organic amino acids such as D-amino acids, with L-amino acids being preferred. The term amino acid further includes salts of the amino acid, such as the sodium salt, or the respective hydrate or hydrochloride salt. For example, tyrosine means L- or D-tyrosine, preferably L-tyrosine, as well as salts or hydrates or hydrochlorides thereof.

[0050] Examples of vitamins are 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), vitamin B12 (cyanocobalamin, hydroxycobalamin, methylcobalamin), vitamin C (ascorbic acid), vitamin D (ergocalciferol, cholecalciferol), vitamin E (tocopherol, tocotrienol), and vitamin K (phylloquinone, menaquinone). Vitamin precursors are also included.

[0051] Examples of salts are compounds containing inorganic ions such as bicarbonate, 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 are copper(II) sulfate pentahydrate (CuSO4·5H2O), sodium chloride (NaCl), calcium chloride (CaCl2·2H2O), potassium chloride (KCl), iron(II) sulfate, ammonium ferric citrate (FAC), anhydrous sodium dihydrogen phosphate (NaH2PO4), anhydrous magnesium sulfate (MgSO4), anhydrous disodium hydrogen phosphate (Na2HPO4), magnesium chloride hexahydrate (MgCl2·6H2O), zinc sulfate heptahydrate. Examples of buffers are CO2 / HCO3 (carbonate), phosphate, HEPES, PIPES, ACES, BES, TES, MOPS, and TRIS.

[0052] Examples of cofactors are thiamine derivatives, biotin, vitamin C, NAD / NADP, cobalamin, flavin mononucleotides and derivatives, glutathione, heme nucleotide phosphates and derivatives. According to the present invention, nucleic acid building blocks 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 or adenosine diphosphate or adenosine triphosphate.

[0053] Feed media may have a different composition compared to complete media. They typically contain amino acids, trace elements, and vitamins. They may also contain a sugar component, although sometimes for production reasons the sugar component is added in a separate feed.

[0054] A suitable feed medium may, for example, contain one or more of the following compounds: L-Asparagine Monohydrate L-Isoleucine L-Phenylalanine Monosodium L-glutamate monohydrate L-Leucine L-Threonine L-Lysine Monohydrochloride L-Proline L-Serine L-Arginine Monohydrochloride L-Histidine Monohydrochloride Monohydrate L-Methionine L-valine Monosodium L-aspartate monohydrate L-tryptophan Choline chloride MYO-inositol Nicotinamide Calcium pantothenate-D(+) Pyridoxine hydrochloride Thiamine chloride hydrochloride Micronized Vitamin B12 (Cyanocobalamin) Biotin folic acid Riboflavin

[0055] Magnesium sulfate anhydrous Copper(II) sulfate pentahydrate Zinc sulfate heptahydrate 1,4-Diaminobutane dihydrochloride Ammonium heptamolybdate tetrahydrate Cadmium sulfate hydrate Manganese(II) chloride tetrahydrate Nickel(II) chloride hexahydrate sodium metasilicate Sodium Metavanadate Tin(II) chloride dihydrate Sodium selenite (approximately 45% SE) Sodium dihydrogen phosphate monohydrate Ammonium iron(III) citrate (approximately 18% FE).

[0056] According to the present invention, freezing means cooling to a temperature below 0°C. In the perfusion process, cell culture medium is continuously added to a bioreactor and removed through a pump, while the cells are retained in the bioreactor by a cell retention device. The advantages of perfusion are the potential to reach very high cell densities (due to regular medium changes) and the potential to produce very fragile recombinant proteins, since the product can be removed from the bioreactor daily, thereby reducing exposure time to high temperatures, redox potential, or released cellular proteases.

[0057] Processes for perfusion cell culture typically involve culturing cells in a bioreactor system comprising a bioreactor with a medium inlet and a harvest outlet, wherein i. During the cell culture process, fresh cell culture medium is continuously or once or several times, preferably continuously, inserted into the bioreactor via the medium inlet. ii. During the cell culture process, continuously or once or several times, preferably continuously, a harvest is removed from the bioreactor via a harvest outlet. The harvest typically includes the desired product produced by the cells, the cells, and the liquid cell culture medium.

[0058] Amino acids are essential components of cell culture media because they are important for supporting cell growth. In addition, amino acids are key building blocks of recombinant proteins produced using mammalian cell culture techniques. Amino acid solubility is a limiting factor that prevents the concentration of cell culture media and feed formulations. Such concentration is essential for developing next-generation manufacturing platforms. In particular, biomanufacturing processes using in-line dilution require highly concentrated formulations to potentially reduce the volume of cell culture media that must be stored in tanks (i.e., reducing the manufacturing footprint) or reduce the volume of feed added, typically through fed-batch processes, thereby increasing volumetric titer.

[0059] It has been found that some amino acids in cell culture media can be replaced with keto acids or their salts. In addition to their use as amino acid sources, the sodium salts of these keto acids, in particular, exhibit high solubility compared to their corresponding amino acids and can therefore be used in highly concentrated formulations. Aside from the advantage of solubility, the use of keto acids has also been found to enable the reduction of ammonia, a known toxic and inhibitory metabolite, in cell culture. Furthermore, the use of certain keto acids has been shown to result in more stable formulations with reduced color change, no or delayed precipitation, and reduced or delayed formation of by-products when stored at room temperature.

[0060] Thus, keto acids of amino acids and their salts can be used in cell culture media formulations for the following applications: Application 1: Increasing total medium / feed solubility Application 2: Replacing the corresponding amino acids and reducing ammonium ion / ammonia preparations in cell culture Application 3: Increase media stability, reduce color change and precipitation due to storage of formulations at 4°C or room temperature, and reduce ammonia formation during feed storage.

[0061] Table 1 shows the amino acids leucine, isoleucine, valine, phenylalanine, and methionine and their corresponding keto acids or sodium salts of the corresponding keto acids. As can be seen from Table 1, the solubility of the corresponding keto acids is higher than that of the amino acids. [Table 1] Table 1. Solubility of amino acids and their respective keto acids or their salts in water at 25° C. Solubility experiments were performed using saturated solutions and residual mass determination after infrared drying.

[0062] It has been found that by partially or totally substituting the amino acids leucine, isoleucine, valine, phenylalanine, and / or methionine with the corresponding keto acids and / or their derivatives, the solubility of a dry powder or dry granule cell culture medium can be improved compared to an otherwise identical cell culture medium without having a negative effect on cell culture performance. In a preferred embodiment, the sodium salts of keto acids are used, as they typically exhibit the highest solubility.

[0063] Suitable derivatives are metal salt derivatives, peptide derivatives, ester derivatives, and other derivatives. The derivatives are keto acid derivatives, which have higher solubility in water compared to the corresponding amino acids, and which can cooperate back into the corresponding amino acids in cells or otherwise replace the corresponding amino acids in their role in maintaining and / or supporting the growth of cells in vitro. Metal salt derivatives are the most preferred derivatives. These are metal salts of keto acids such as sodium, potassium, calcium, or magnesium salts, preferably sodium salts.

[0064] A peptide derivative is one in which one or more, typically one, two or three, amino acids are linked to a keto acid via peptide bonds. In this case of ketroucine, the structural formula of a peptide derivative is shown in Scheme 1 below: [ka] R 1 is the amino acid side chain, and R 2 is another amino acid linked via a peptide bond.

[0065] The ester derivative is a derivative in which the carboxylic acid of a keto acid forms an alkyl or aryl ester. Most preferably, it is a C1-C4 alkyl ester. An example of a keto-leucine ester derivative is shown in Scheme 2: [ka] R 2 is alkyl or aryl, and the alkyl group is -OH or OR 2 or may form, for example, an ether or ester.

[0066] Suitable R 2 Examples of are methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, benzyl and [ka] is.

[0067] Other derivatives are shown in Scheme 3: [ka]

[0068] The above example given for ketroucine can of course be equally realised for other keto acids from the group 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid.

[0069] The present invention is therefore directed to a dry powder or dry granule cell culture medium comprising at least one alpha keto acid from the group consisting of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid, and alpha-keto gamma methylthiobutyric acid and / or their derivatives, preferably a metal salt derivative, most preferably a sodium salt. In a preferred embodiment, the dry powder or dry granule cell culture medium comprises the sodium salts of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, and / or alpha-ketoisovaleric acid, most preferably the sodium salts of all three keto acids.

[0070] The amount of keto acids in the dry powder or dry granule cell culture medium is such that the concentration of each keto acid and / or its derivative in the liquid medium obtained after dissolution of the dry powder or dry granule cell culture medium is greater than 10 mM, preferably 20 to 600 mM, and most preferably 30 to 300 mM.

[0071] In one embodiment, the dry powder or dry granule cell culture medium containing a keto acid as defined above does not contain the corresponding amino acid. In another embodiment, the dry powder or dry granule cell culture medium containing a keto acid as defined above contains up to 50% (mol%) of the corresponding amino acid. For use of dry powder or dry granular media, a solvent, preferably water (most particularly distilled and / or deionized water, also purified water or water for injection) or an aqueous buffer, is added to the media and the components are mixed until the media is completely dissolved in the solvent.

[0072] The solvent may also include saline, soluble acid or base ions to provide a suitable pH range (typically between pH 1.0 and pH 10.0, preferably between 6.5 and 8.5), stabilizers, surfactants, preservatives, and alcohol or other polar organic solvents. The mixture of cell culture medium and solvent may also contain additional substances such as buffer substances for pH adjustment, fetal bovine serum, sugars, etc. The resulting liquid cell culture medium is then contacted with the cells to be grown or maintained.

[0073] Dry powder or dry granule medium compositions containing higher concentrations of leucine, isoleucine, valine, phenylalanine and methionine exhibit turbidity when mixed with solvents due to the limited solubility of the amino acids, whereas cell culture media according to the present invention using the same concentrations of the corresponding keto acids and / or their derivatives give clear solutions, which are particularly suitable for feed media.

[0074] The resulting liquid medium containing keto acids and / or their derivatives exhibits at least the same performance in cell culture. It has been found that amino acids can be completely replaced with the corresponding keto acids and / or derivatives, preferably their salts. Nevertheless, it is also possible to only partially replace amino acids. In this case, preferably 50% (mol%) or more of the amino acids are replaced with the corresponding keto acids and / or their derivatives.

[0075] In some cases, it may be advantageous to modify, particularly increase, the amount of keto acid relative to the amount of the amino acid being substituted. For isoleucine, leucine, and valine substitutions, a 1:1 substitution is typically sufficient, while for phenylalanine and methionine, it has been found that it is typically preferable to add a larger amount of keto acid relative to the amount of amino acid. Typically, a substitution of 1:1.1 to 1:3 (molar basis) is preferred.

[0076] Preferably, the maximum solubility of the medium can be increased by completely replacing the amino acids leucine, isoleucine, valine, phenylalanine, and methionine with the corresponding keto acids and / or their derivatives, especially with the sodium salts of keto acids. As can be seen in Example 3, the solubility of the dry powder medium can be doubled, for example. In addition to improving the solubility of dry powder or dry granule media by substituting amino acids as described above, it has also been unexpectedly found that the specific productivity of cell cultures is increased when media are used in which leucine and / or isoleucine are replaced with the corresponding keto acids and / or their salts.

[0077] It was further demonstrated that three critical quality attributes of IgG1 produced using media in which leucine and / or isoleucine were replaced with the corresponding keto acids and / or their salts were not different between the control and substituted amino acid conditions: glycosylation pattern, antibody aggregation and fragmentation, and charge variants.

[0078] It has further been found that keto acids and / or their derivatives, in particular keto acids and / or salts of leucine, isoleucine and valine, preferably 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid and / or their salts, are suitable for stabilizing liquid cell culture medium formulations. Liquid media containing the components exhibit less color change when stored at either room temperature or 4°C over a period of 3 months, with or without light exposure, compared to media lacking 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid and / or their salts but containing the same amount of the corresponding amino acid. They also exhibited reduced precipitation.

[0079] This effect can be achieved by replacing the corresponding amino acids, such as isoleucine and / or leucine, with the corresponding keto acids and / or their derivatives. This effect can also be achieved by adding the corresponding keto acids, such as 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, and / or their derivatives, to a cell culture medium formulation containing leucine and / or isoleucine. This means that keto acids and / or their derivatives can be used as medium stabilizers regardless of the medium composition. The preferred concentration in a liquid formulation is at least 20 mM, preferably 30-600 mM.

[0080] The powdered cell culture medium of the present invention is preferably produced by mixing all components and grinding them. Mixing components is well known to those skilled in the art of producing dry powdered cell culture media by grinding. Preferably, all components are thoroughly mixed so that all parts of the mixture have nearly the same composition. For uniform cell growth, the more uniform the composition, the better the quality of the resulting medium. The grinding can be carried out by any type of mill suitable for the production of cell culture media. Typical examples are a ball mill, a pin mill, a Fitz mill, or a jet mill. Preferably, it is a pin mill, a Fitz mill, or a jet mill, and most preferably a pin mill. Those skilled in the art know how to implement such a mill.

[0081] Large scale equipment mills with disc diameters of about 40 cm, for example pin mills, typically operate at 1 to 6500 revolutions per minute, preferably 1 to 3000 revolutions per minute. Milling can be carried out under standard milling conditions and results in a powder with a particle size between 10 and 300 μm, most preferably between 25 and 120 μm.

[0082] Particle size refers to the diameter of the particle. Particle diameter is determined by laser light scattering. Using this technique, particle size is reported as the volume-equivalent sphere diameter. The particle size range refers to the particle size range of 75% or more, preferably 90% or more of the particles, which means that if the particle size is 25 to 120 μm, at least 75% of the particles have a particle size of 25 to 120 μm.

[0083] Preferably, all components of the mixture subjected to milling are dry, meaning that if they contain water, they contain no more than 10%, preferably no more than 5%, and most preferably no more than 2% water crystals by weight of unbound or ungrouped water molecules. In a preferred embodiment, the grinding is carried out in an inert atmosphere, the preferred inert protective gas being nitrogen.

[0084] In a further preferred embodiment, all components of the mixture are frozen prior to grinding. Freezing of the components prior to grinding can be done by any means that ensures cooling of the components to a temperature below 0°C, and most preferably below -20°C. In a preferred embodiment, freezing is done by liquid nitrogen. This means that the components are treated with liquid nitrogen, for example by pouring liquid nitrogen into a container in which the components are stored, prior to introduction into the mill. In a preferred embodiment, the container is a feeder. If the container is a feeder, preferably the liquid nitrogen is introduced near or adjacent to the feeder into which the components are introduced.

[0085] Typically, the components are treated with liquid nitrogen for 2 to 20 seconds. Preferably, the cooling of the ingredients is done in such a way that all ingredients entering the mill are at a temperature below 0°C, most preferably below -20°C.

[0086] In a preferred embodiment, all ingredients are placed in a container from which the mixture is transferred into a feeder, most preferably a metering screw feeder. In the feeder, the ingredients are sometimes further mixed—depending on the type of feeder—and additionally cooled. The frozen mixture is transferred from the feeder to the mill so that the mixture to be ground in the mill still has a temperature preferably below 0° C., more preferably below −20° C.

[0087] Typically, the mixing time (meaning the residence time of the mixture of ingredients in the feeder) is more than 1 minute, preferably between 15 and 60 minutes. Metering screw feeders (also called lightweight snails) typically run at speeds of 10 to 200 revolutions per minute, preferably 40 to 60 revolutions per minute.

[0088] Typically, the grinding temperature is kept between -50 and +30°C. In a preferred embodiment, the temperature is kept at approximately 10°C. The oxygen level during milling is preferably less than 10% (v / v). The process can be carried out, for example, batchwise or continuously, In a preferred embodiment, the process according to the present invention is made continuous by permanently filling a feeder with a mixture of ingredients for cooling over a period of time, and then permanently filling the mill with the cooled mixture from the feeder.

[0089] The present invention provides a) Providing a bioreactor b) providing a liquid cell culture medium comprising at least one alpha-keto acid from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid and / or derivatives thereof, preferably at a concentration of more than 10 mM; c) mixing the cells to be cultured with a liquid cell culture medium; d) incubating the mixture of step b). The present invention is further directed to a process for culturing cells according to the present invention.

[0090] In a preferred embodiment, the cells are CHO cells. In one embodiment, the liquid cell culture medium provided in step b) is a liquid cell culture medium in which one or more of the amino acids isoleucine, leucine, valine, phenylalanine and methionine are partially or preferably completely substituted with a corresponding keto acid selected from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid and / or derivatives thereof.

[0091] In a preferred embodiment, the liquid cell culture medium of step b) is provided by dissolving a dry powder or dry granular medium according to the invention in a solvent as described above. A bioreactor is any vessel or tank in which cells can be cultured. The culture is typically under appropriate conditions, such as an appropriate temperature. Those skilled in the art will know the appropriate incubation conditions to support or maintain cell growth / culture.

[0092] The present invention has been found to be highly suitable for the preparation of feed media, the concentration of which is limited due to solubility issues, especially due to the limited availability of certain amino acids at the concentrations required for the feed medium.

[0093] As a result, there is a need for a feed medium that contains all the required components at high concentrations in one feed. Additionally, the pH of the feed should not negatively affect the cell culture, i.e., the pH of a liquid feed should be less than 8.5, preferably between 6.5 and 7.8.

[0094] It has been found that partial or preferably complete replacement of the amino acids isoleucine, leucine, valine, phenylalanine, and methionine with the corresponding keto acids and / or derivatives, preferably their salts, improves the solubility of the resulting dry powder medium. This offers the possibility of producing liquid media containing higher concentrations of components, so that the same amount of components can be added to cell culture in a smaller amount of liquid, yet at a suitable pH, preferably below 8.5. More highly concentrated feed media containing keto acids can be used without any negative, and in some cases even with positive, effects on cell growth and / or productivity and liquid medium stability.

[0095] Thus, the present invention is also directed to a feed medium, either in the form of a powder medium or after dissolution in the form of a liquid medium. The resulting liquid medium contains at least one keto acid selected from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid and / or their derivatives at a concentration of more than 10 mM, preferably 20-600 mM, and preferably has a pH of 8.5 or less. In a preferred embodiment, the pH is 6.7 to 8.4.

[0096] The present invention also provides - loading the cells and aqueous cell culture medium into the bioreactor - Incubating the cells in a bioreactor - adding cell culture medium, in this case a feed medium, to the bioreactor continuously throughout the incubation of the cells in the bioreactor or once or several times during said incubation period; wherein the feed medium preferably has a pH of less than pH 8.5 and comprises at least one keto acid selected from the group consisting of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid and / or derivatives thereof, This paper is directed to a fed-batch process for culturing cells in a bioreactor.

[0097] Preferably, the feed medium contains one or more keto acids and / or their derivatives at a concentration of more than 10 mM, preferably 20-600 mM. Preferably, the feed medium contains 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid and / or alpha-ketoisovaleric acid and / or their salts, most preferably the sodium salts. Typically, the feed medium contains 50-400 g / L of solid components dissolved in a solvent.

[0098] In a preferred embodiment, in the process of the present invention, the feed medium that is added to the bioreactor continuously during incubation or once or several times during said time always has the same composition. In a preferred embodiment, the cells are CHO cells. The present invention is further illustrated by the following figures and examples, but is not limited thereto. The entire disclosures of all applications, patents, and publications, cited above and below, are hereby incorporated by reference.

[0099] example The following examples illustrate practical applications of the present invention. Example 1: Keto acids have increased solubility in water compared to their respective amino acids The maximum solubility of five exemplary amino acids was compared by preparing saturated solutions of each keto acid or its salt in water at 25° C. After precipitation, the solutions were dried using infrared light (120° C., 120 min) and the residual mass determined in g / kg. As shown in Figure 1, the solubility of keto acids and their salts was significantly higher than that of the respective amino acids in water. To rule out that the increased solubility was due to the sodium salt forms of the keto acids, a separate experiment was performed to compare the solubilities of Leu, Leu sodium salt, and keto-Leu sodium salt. The maximum solubilities obtained in water were 22.1, 86.0, and 313.7 g / kg, respectively. As expected, the formation of the sodium salt already increased the solubility of Leu, but the increase in solubility obtained with the keto acids was significantly more significant and therefore not due solely to the salt form.

[0100] Example 2: Maximum solubility of keto acids compared to their respective amino acids in Ile and Leu depleted 4 Feeds Increasing amounts of keto acids and their salts were added to a cell culture feed formulation (Cellvento® 4Feed, MilliporeSigma) depleted of Ile and Leu. Similarly, as a control, increasing amounts of Ile and Leu were added to the same feed formulation. The total concentration of this feed formulation was 125 g / L, and the pH was 7.0 + / - 0.2. In small-scale experiments, after each addition of either amino acids or keto acids, the feed was stirred for 10 minutes and the turbidity was measured. The experiments were performed at room temperature (25°C).

[0101] The maximum solubility of Ile in Cellvento® 4Feed depleted of Ile / Leu was approximately 105 mM, while keto-Ile was found to be soluble at the highest tested concentration of 635 mM with turbidity values ​​of less than 5 NTU (see Figure 1 ), indicating that keto-Ile is at least six times more soluble than Ile in 4Feed depleted of Ile / Leu. The maximum solubility of Leu in Cellvento® 4Feed depleted of Ile and Leu was approximately 90 mM, while for keto-Leu, the maximum soluble concentration (turbidity value less than 5 NTU) was found to be 240 mM (see Figure 2), indicating that keto-Leu is 2.6 times more soluble than Leu in 4Feed depleted of Ile / Leu.

[0102] Example 3: The use of keto acids allows for the concentration of cell culture media formulations at neutral pH. The maximum solubility of Cellvento® 4Feed was determined by dissolving increasing amounts of feed dry powder medium in water until precipitation was visually detected. For each condition, the feed was stirred for approximately 30 minutes, the pH was adjusted to 7.0 + / - 0.2, and the solution was stirred for an additional 10 minutes for equilibration. Osmolality and turbidity were measured (see Figure 3). The data show that a 1.2x concentrate of this formulation already does not dissolve, as particles are detected in suspension and turbidity is well above the 5 NTU limit.

[0103] Because Ile and Leu were identified as the primary limiting amino acids for enrichment of the Cellvento® 4Feed formulation, a new backbone feed lacking Ile and Leu was produced (4Feed-Ile / Leu). The maximum concentration of this feed, with or without supplementation with keto-Leu and keto-Ile, was determined by dissolving increasing amounts of feed dry powder medium in water until precipitation was visually detected. For each condition, the feed was stirred for approximately 30 minutes, the pH was adjusted to 7.0 + / - 0.2, and the solution was stirred for an additional 10 minutes for equilibration. Turbidity was measured, and a limit of 5 NTU was considered soluble.

[0104] The results show that the maximum solubility of Ile / Leu-deficient Cellvento® 4 Feed was approximately 228 g / L. When keto-Leu and keto-Ile were added, maximum solubility was obtained at 216 g / L to 228 g / L of depleted dry powder medium supplemented with a combined amount of keto-Leu and keto-Ile of 36 g / L to 38 g / L (a molar ratio equivalent to the theoretical amount of Ile and Leu in the concentrate), resulting in a total concentration of 252 g / L to 266 g / L for the formulation containing both keto-Leu and keto-Ile. Considering that Cellvento® 4 Feed has a concentration of 130 g / L, this represents a 100% increase in concentration when Ile and Leu are replaced with keto-Ile and keto-Leu. The data show that it is possible to concentrate the formulation at least 2x (265 g / L) since no particles can be detected in the suspension and the turbidity is less than 5 NTU (see Figure 4).

[0105] Example 4: Keto acids of Leu and Ile can stabilize cell culture media formulations The stability of a feed containing Ile and Leu (Cellvento® 4Feed) was compared to that of the same feed depleted of Ile / Leu and supplemented with either keto-Leu or keto-Ile. The feed was prepared according to standard protocols. The final pH was 7.0 ± 0.2, and the feed was stored at 4°C or RT, protected from light or exposed to light. The color change of the formulation was monitored over 90 days by measuring absorbance in the range 300 nm to 600 nm (5 nm intervals). Conditions were compared by calculating the baseline-corrected area under the curve (AUC) of the absorbance scan (300 nm to 600 nm) over time (between D0 and D90).

[0106] As shown in Figure 5A, the feed containing Ile and Leu in the control condition darkened in color (AUC increased from 350 to 7000) with increasing temperature or light exposure. At 4°C, when Leu was replaced with keto-Leu, the AUC decreased by 27% and 8% in the light-protected and light-exposed conditions, respectively. At RT, the decrease was even more pronounced, with the AUC decreasing by 31% (light-protected) and 37% (light-exposed) in the keto-Leu condition, respectively. This indicates that replacing Leu with keto-Leu can significantly reduce the color change observed in the feed over time.

[0107] The results obtained for keto-Ile are presented in Figure 5B. As with keto-Leu, a decrease in AUC was observed when Ile was replaced with keto-Ile. At 4°C, AUC decreased by 33% and 68% in the light-protected and light-exposed conditions, respectively. At RT, no decrease was observed in the light-protected condition, but a 38% decrease was observed in the light-exposed condition, indicating that replacing Ile with keto-Ile can significantly reduce the color change observed in the feed over time. Overall our results show that replacement of amino acids with their keto acids or their salts can result in stabilization with less color change when stored for 3 months at either 4 °C or RT, with or without light exposure.

[0108] In addition, when keto-Leu was used in place of Leu in the feed, precipitation of the feed was delayed. To observe precipitation, 50 mL Falcon tubes were inverted to check for any precipitate at the bottom of the tube and photographed. At 4°C, no precipitation occurred under any condition. However, under RT light protection, precipitation occurred between D49 and D70 in the control condition, while no precipitation was observed in the keto-Leu-containing condition. While complete inhibition of precipitation was not observed under RT light exposure, precipitation was delayed in the keto-Leu condition. Precipitation was observed from D49 in the control condition, while the first precipitate appeared at D70 in the keto-Leu condition. Over the next few days, the amount and color intensity of the precipitate decreased in the keto-Leu-containing condition, indicating a slight increase in the stability of the keto-Leu formulation, even under RT light exposure.

[0109] Finally, the amount of ammonium ion formed during storage at 4°C or RT for the keto acid-containing feeds was less when compared to the regular amino acid-containing feeds. To allow for assessment of NH3 formation over the course of the stability study, the AUC of NH3 concentration was calculated over a 3-month timeframe and conditions were compared. The results for keto-Leu are presented in Figure 6A and show less ammonia formation compared to the control condition. When the feed was stored at 4°C with light protection and light exposure, 10% and 19% less ammonia was produced in the keto-Leu condition compared to the control, respectively. At RT, the same trend was observed; ammonia levels decreased by 15% and 5%, respectively, when the feed was stored with light protection and light exposure for 3 months.

[0110] Similar results were obtained for ketoIle (Figure 6B), showing less ammonia formation when compared to the control condition. When the feed was stored at 4°C with light protection and light exposure, ammonia was produced 21 and 24% less in the ketoIle condition compared to the control, respectively. At RT, the same trend was observed, with ammonia levels decreasing by 28 and 25%, respectively, when the feed was stored with light protection and light exposure for 3 months.

[0111] Example 5: Keto-Ile and keto-Leu can replace their respective amino acids in the feed to increase specific productivity. Cell culture results with an IgG1-producing CHOK1GS clone. For cell culture experiments, the CHOK1GS suspension cell line expressing human IgG1 was used. Cells were cultured in a starting culture volume of 30 mL and 2x10 5 The cells were cultured in quadruplicate in Cellvento 4CHO medium (Merck Darmstadt, Germany) in 50 mL spin tubes at a seeding density of 1000 cells / mL. Incubation was carried out at 37°C, 5% CO2, 80% humidity, and 320 rpm agitation. Keto acids were added to the feeds (Feed 4 lacking Ile and Leu) in place of their respective amino acids. The pH of all feeds was neutral (pH 7.0 ± 0.2). Positive controls contained normal amino acids, while negative controls contained feeds lacking the respective amino acids without added keto acids. Feeds were performed at the following v / v ratios (3, 3, 6, 3, and 3%) on days 3, 5, 7, 10, and 14. Glucose was determined daily and adjusted to 6 g / L using a 400 g / L glucose solution. Experiments were repeated at least three times.

[0112] Viable cell density (VCD) and viability were assessed using a Vi-CELL XR (Beckman Coulter, Fullerton, CA). Metabolite concentrations were monitored spectrophotometrically and turbidimetrically using a Cedex Bio HT (Roche Diagnostics, Mannheim, Germany). Amino acid quantification was performed by UPLC after derivatization using the AccQ·TagUltra® reagent kit. Derivatization, chromatography, and data analysis were performed according to the supplier's recommendations (Waters, Milford, MA).

[0113] Productivity per cell per day was calculated each day by dividing the titer by the corrected integral VCD to account for dilution caused by feeding. Overall specific productivity was determined by calculating the slope from the linear regression between titer and the corrected integral VCD. Looking at viable cell density (Figure 7A), both keto derivatives led to a slightly lower maximum VCD compared to the control, but the titer obtained after 11 days (Figure 7B) was slightly higher than the control condition, indicating an overall higher specific productivity (Figure 8). The negative control, in which the feed was deficient in Leu and Ile, showed a sharply reduced VCD after 7 days and, most importantly, a very limited IgG titer, indicating that Leu and Ile are important for supporting IgG production by CHO cells.

[0114] NH is an undesirable metabolite produced over the course of the fed-batch process. The amount of NH produced during the 17-day fed-batch process in the keto-Leu and keto-Ile conditions (FIG. 9A) was significantly reduced compared to the control containing Leu and Ile, indicating either that a significant portion of the ammonia is generated from the oxidative deamination of Leu and Ile, or that the presence of keto acids in the bioreactor medium promotes the utilization of free NH as a building block for producing amino acids by amination.

[0115] The concentrations of amino acids in the spent medium were determined. In the condition where Leu was replaced with keto-Leu, the concentration of Leu in the spent medium (Figure 9B) was slightly lower than that of the positive control (containing Leu and Ile). However, the evolution over time showed an increase in concentration between the feeding day and the next day, indicating that Leu can be produced very rapidly from keto-Leu. In the condition where Ile was replaced with keto-Ile (Figure 10A), the concentration of Ile detected over time was significantly lower than that in the positive control, indicating either a slow conversion of keto-Ile to Ile or the formation of another product from keto-Ile during cultivation. Comparison of the keto-Ile condition with the negative control, in which the feed was deficient in Ile and Leu, showed that Ile could nevertheless be produced from keto-Ile in the fed batch. In addition, careful analysis of the chromatograms allowed the identification of a new peak corresponding to allo-Ile, which increased over time (Figure 10B).

[0116] The quality of antibody produced in a control fed-batch process (feed containing Ile and Leu) was compared to the quality of antibody produced in feeds depleted of either Leu and Ile and supplemented with either keto-Leu or keto-Ile. Antibodies were purified from cell culture supernatants using Protein A PhyTips® (PhyNexus Inc, San Jose, CA). After derivatization using the GlykoPrep®-plus Rapid N-Glycan Sample Preparation kit with 8-aminopyrene-1,3,6-trisulfonic acid trisodium (APTS) (Prozyme, Hayward, CA) according to the manufacturer's instructions, glycosylation patterns were analyzed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF). Briefly, purified antibodies were denatured and fixed, digested with N-Glycanase®, and then labeled with APTS at 50°C for 60 minutes to release glycans from the antibodies. After a wash step to remove residual APTS, the relative amounts of glycans were determined using a Pharmaceutical Analysis System CESI8000 Plus (Sciex, Washington, USA) equipped with an LIF detector (Ex: 488 nm, Em: 520 nm).

[0117] Separation was performed in a polyvinyl alcohol-coated capillary (total length: 50.2 cm, inner diameter: 50 μm) filled with carbohydrate separation buffer from a carbohydrate labeling kit (Beckman Coulter, Brea, USA). The capillary surface was first rinsed with separation buffer at 30 psi for 3 min. The inlet and outlet buffer vials were replaced every 20 cycles. Samples were introduced by pressure injection at 0.5 psi for 12 s, followed by a 0.2 min soak step to clean the capillary tip. Finally, separation was performed at 20 kV for 20 min with reverse polarity applied, with a 0.17 min ramp. Peaks were identified according to their individual migration times and integrated according to the following parameters: peak width 0.05, threshold 10,000, and shoulder sensitivity 9,999.

[0118] Antibody aggregation and fragmentation were measured using size exclusion chromatography on a Water Acquity UPLC system using a TSKgel SuperSW3000 column (Tosoh Bioscience). The mobile phase was 0.05 M sodium phosphate, 0.4 M sodium perchlorate, pH 6.3, with a flow rate of 0.35 mL / min. Sample concentrations were adjusted to 1.0 mg / mL using storage buffer after IgG purification, and detection was performed using absorbance at 214 nm.

[0119] Charge variants were measured using cIEF on a Capillary Electrophoresis CESI 8000 (Beckman Coulter / Sciex) according to the manufacturer's instructions. Sample concentrations were adjusted to 1.5 mg / mL using storage buffer after IgG purification. Prior to measurement, samples were mixed with a master mix containing various pH markers, cathode / anodic stabilizers, 3M urea cIEF gel, and Pharmalyte. The results obtained for glycosylation (Figure 11), high and low molecular weight species (Figure 12A), and charge variants (Figure 12B) show no differences between the control conditions and the conditions in which Ile and Leu were exchanged with ketoIle and ketoLeu, indicating that the amino acid exchanges do not affect the three important quality attributes of the IgG1 produced in this study.

[0120] Example 6: Confirmation of keto-Leu performance using IgG1-producing CHODG44 and CHOK1 clones. The applicability of the present technology to various bioprocesses was demonstrated by conducting fed-batch experiments using other types of CHO cells, such as CHODG44 and CHOK1 (non-GS), with keto-Leu. Results for the DG44 cell line ( FIG. 13 ) show that the VCD was lower and the IgG titer was slightly lower under keto-Leu conditions compared to the control. Nevertheless, the process using keto-Leu slightly increased the overall specific productivity. Spent medium data showed that the Leu concentration under keto-Leu conditions was similar to that of the control for this cell line, confirming that Leu can also be produced very rapidly from keto-Leu in this cell line. Figure 13: Performance of keto-Leu-containing processes in the IgG1-expressing CHODG44 cell line compared to the control. Figure 14: Performance of keto-Leu-containing processes in the IgG1-expressing CHOK1 non-GS cell line compared to the control.

[0121] Example 7: Performance in batches with different seeding densities and different leu / ketoLeu ratios Our fed-batch spent medium results obtained with three different CHO cell lines using keto-Ile and keto-Leu show that the formation of Ile, allo-Ile, and Leu from keto acids is very rapid. This indicates that, because keto acids are readily available from the beginning of the culture, they may also be used to increase the solubility of batch and perfusion media. To confirm this applicability in the CHO system, keto-Leu or keto-Ile were used as substitutes for Leu and Ile, respectively, in cell culture medium (Cellvento® 4CHO). Leu / Ile-deficient versions of the formulation were produced, using equimolar concentrations of keto-Leu to Leu and keto-Ile to Ile (Figure 15). Cell growth and viability of the CHOK1GS cell line were monitored over several weeks in serial passage experiments to confirm that growth was not due to residual amounts of Leu or Ile. Batch experiments were designed at a seeding density of 0.2 million cells / mL, and IgG production was measured over time. Quantification of amino acids in spent medium was also used to follow amino acid production over time.

[0122] In a similar manner, batch experiments at higher cell seeding densities were performed using media containing various Leu / ketoLeu ratios to understand which ratios were preferred when starting at higher cell densities (Figure 16). The analytical method used was the same as described above. The results of serial passaging indicate that CHOK1GS cells cannot grow in media lacking Ile and Leu, as no growth was observed during the first few days of culture and viability was significantly reduced. In contrast, continuous growth was observed when Leu or Ile was replaced with their corresponding ketoacids. Overall, the maximum viable cell densities observed at each passage were slightly lower than in the control conditions containing Ile and Leu, indicating that smaller amounts of Leu and Ile are required to achieve performance comparable to that of the control conditions. This amount can be quite easily determined experimentally by testing media containing various ratios of Ile / Leu and ketoIle / ketoLeu.

[0123] In batch experiments, performance was comparable between the control and keto-Leu conditions, indicating that CHOK1GS cells could grow when Leu was replaced with a molar equivalent of keto-Leu (Figure 15A). Under these conditions, similar amounts of IgG were detected on days 7 and 10 (Figure 15B). In contrast, when Ile was replaced with keto-Ile, growth and IgG concentrations after day 5 were slightly impaired, suggesting that a smaller amount of Ile may be required to achieve growth and titers similar to those of the batch control over time. This amount can be determined empirically quite easily by testing media containing various ratios of Ile and keto-Ile. Alternatively, higher molar keto-Ile concentrations compared to Ile concentrations can be tested.

[0124] The difference in performance between keto-Ile and keto-Leu may be explained by looking at the formation of Ile, allo-Ile, and Leu in the spent medium. While 34% of the initial keto-Leu concentration was detected in the form of Leu on day 3, only 21% of the initial keto-Ile concentration was detected as Ile on day 3. In addition, 35% of the initial keto-Ile concentration was detected in the form of allo-Ile by day 10. This indicates that the amination of keto-Leu to Leu by cells is more efficient than the amination of keto-Ile to Ile due to the concomitant formation of allo-Ile, which may not be used to the same extent as Ile by cells.

[0125] Finally, batch experiments at higher cell densities were performed to determine whether keto-Leu is readily available when starting at high seeding densities, or whether a minimum concentration of free leucine must be present to support growth and productivity in these conditions. In these experiments, the CHOK1GS cell line was seeded at 0.3, 0.6, or 1.10^6 cells / mL in medium containing 0, 25, 50, 75, or 100% keto-Leu, with the remainder added in the form of leucine.

[0126] The results show, as expected, increased growth and titer with increasing seeding density. Among the various keto-Leu / Leu ratios, the greatest VCD was observed at the highest seeding density of 1.10^6 cells / mL, with 100% keto-Leu exchange. When cells were seeded at 0.6.10^6 cells / mL and 0.3.10^6 cells / mL, the highest VCD was observed at a 1:1 keto-Leu:Leu ratio (50% keto-Leu, 50% Leu). While no significant differences in titer were observed between seedings at 0.3 and 1.10^6 cells / mL, there was a slight trend toward increased IgG concentration with higher Leu / keto-Leu ratios at 0.6.10^6 cells / mL. This difference may not be significant.

[0127] Example 8. Performance of other keto acids versus their respective amino acids in FB cultures In the FB experiments, other keto acids were tested as substitutes for their respective amino acids. When Val was replaced with ketoVal in the feed, very similar behavior was observed compared to Ile and Leu (Figure 17). Indeed, while similar VCD and titer were observed compared to the positive control, a Val-deficient feed led to a significantly reduced VCD and very low titer after 7 days. NH3 concentrations were also lower when keto acids were used, indicating that utilization of each keto acid, even for Val, can lead to less NH3 during fed-batch cultivation. Overall, this indicates that ketoVal, like members of the branched-chain keto acid family, is likely to behave similarly to ketoLeu and ketoIle and be aminated very rapidly during cell culture. Due to its structural similarity to ketoIle and ketoLeu, the effect of ketoVal on overall feed concentration and feed stability is similar to other branched-chain keto acids. Compared to Val, it exhibited a six-fold higher solubility in water.

[0128] For phenylalanine (Phe) and its keto acid, phenylpyruvate (Figure 18), the amination reaction to produce Phe in cell culture appears to be slower than the amination reaction occurring with branched-chain keto acids. Indeed, when Phe was replaced with an equimolar concentration of phenylpyruvate, spent medium data revealed that although more Phe was found in the supernatant compared to the negative control (feed lacking Phe), the amount formed was not sufficient to support the same growth and titer as in the control condition. After 5 days, a significantly reduced VCD was observed, with a final drop in titer of 20%. This result was followed by a condition in which 2x the molar equivalent of Phe was used as the phenylpyruvate concentration in the feed. The results show that increasing the amount of phenylpyruvate was able to restore similar VCD, titer, and amounts of Phe in the spent medium. These data confirm that Phe can also be replaced with its keto acid, although adjustments to the concentration may be necessary to accommodate the slower pace of the amination reaction.

Claims

1. A dry powder or dry granule cell culture medium comprising at least one alpha-keto acid from the group consisting of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid and / or derivatives thereof.

2. 2. The dry powder or dry granule cell culture medium of claim 1, wherein one or more alpha keto acids from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha keto gamma methylthiobutyric acid and / or derivatives thereof are present in an amount such that, for each of the alpha keto acids, the concentration in the liquid medium obtained after dissolution of the dry powder or dry granule cell culture medium is greater than 10 mM.

3. 3. The dry powder or dry granule cell culture medium according to claim 1 or 2, wherein the dry powder or dry granule cell culture medium does not contain a corresponding amino acid.

4. 4. The dry powder or dry granule cell culture medium of claim 1, wherein the medium comprises sodium salts of one or more alpha keto acids from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid, and alpha keto gamma methylthiobutyric acid.

5. 5. The dry powder or dry granule cell culture medium according to any one of claims 1 to 4, wherein the dry powder or dry granule cell culture medium comprises one or more sodium salts of alpha keto acids selected from 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid and / or alpha-ketoisovaleric acid.

6. a) mixing at least one alpha keto acid from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha keto gamma methylthiobutyric acid and / or derivatives thereof with other components of a cell culture medium; b) subjecting the mixture of step a) to grinding.

6. A method for producing the dry powder cell culture medium of claim 1, by

7. a) Providing a bioreactor b) mixing the cells to be cultured with a liquid cell culture medium prepared by dissolving the dry powder or dry granule medium according to any one of claims 1 to 5 in a solvent; c) incubating the mixture of step b). A process for culturing cells according to.

8. - loading the cells and aqueous cell culture medium into the bioreactor - Incubating the cells in a bioreactor - adding cell culture medium, in this case feed medium, to the bioreactor continuously throughout the incubation of the cells in the bioreactor or once or several times during said incubation period; wherein the feed medium is prepared by dissolving the dry powder or dry granule medium according to any one of claims 1 to 5 in a solvent. A fed-batch process for culturing cells in a bioreactor, according to

9. 9. The fed-batch process of claim 8, wherein the feed medium comprises at least 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid and / or salts thereof in a concentration of 12 to 600 mmol / l.

10. 1. A method for stabilizing a liquid cell culture medium comprising at least 20 mM 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and / or alpha-keto gamma methylthiobutyric acid and / or derivatives thereof in the medium, wherein the resulting medium exhibits less color change and / or less precipitation when stored at 4° C. or at room temperature for 90 days compared to a medium of otherwise identical composition lacking 4-methyl-2-oxopentanoic acid and / or 3-methyl-2-oxopentanoic acid and / or derivatives thereof or in which 4-methyl-2-oxopentanoic acid and / or 3-methyl-2-oxopentanoic acid and / or derivatives thereof are substituted with the corresponding amino acids and / or derivatives thereof.

11. 1. A method for improving the solubility of a dry powder or dry granule cell culture medium by completely or partially replacing one or more of the amino acids isoleucine, leucine, valine, phenylalanine and methionine with the corresponding keto acid selected from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-keto gamma methylthiobutyric acid and / or derivatives thereof.

12. 12. The method of claim 11, by substituting at least 50% (molar ratio) of each amino acid with the corresponding alpha keto acid and / or their derivatives.

13. 13. The method of claim 11 or 12, comprising providing a dry powder or dry granule cell culture medium in which, compared to the original composition, at least 50% (molar ratio) of each amino acid has been substituted with the corresponding alpha-keto acid and / or their derivatives, and dissolving the medium in a solvent, wherein dissolution occurs faster and / or with less solvent than a medium of otherwise identical original composition in which the amino acids have not been substituted.

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