Cell culture medium containing keto acid

Abstract

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.

Description

Technical Field

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

Background Art

[0002] Cell culture media assist and maintain the growth of cells in an artificial environment. Depending on the type of organism whose growth is assisted, cell culture media are complex mixtures of components, sometimes containing over 100 different components. Cell culture media required for the growth of mammalian, insect or plant cells are typically much more complex than media for assisting the growth of bacteria and yeast.

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

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

[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 the increasing market demands and reduce manufacturing costs. To achieve maximum production capacity, in addition to the use of high-performance recombinant cell lines, improvements in cell culture media and process parameters are required.

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

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

[0008] However, in other cell culture processes such as batch processes or perfusion processes, precisely formulated and often highly concentrated medium formulations are required. Especially in perfusion processes, the periodic replacement of the medium in the bioreactor requires the operator to prepare and handle large volumes of liquid medium. To reduce the footprint required to store these volumes, concentration of the medium is required.

[0009] Limitations for the preparation of cell culture media from dry powders are the poor solubility or stability of some components, especially some amino acids. As a result, it is preferable to find a method for providing a dry powder medium composition that is sufficiently soluble to produce a highly concentrated liquid medium composition. It has been found that instead of the amino acids isoleucine, leucine, valine, phenylalanine, and methionine, the respective alpha-keto acids can be used without any negative effects and, in some cases, even with positive effects on cell growth and improved solubility. Furthermore, it has been found that these keto acids even have the effect of stabilizing liquid cell culture medium formulations.

[0010] In a 1959 paper dealing with amino acid metabolism, it was stated that several amino acids could be replaced with their keto acids (Eagle H: Amino acid metabolism in mammalian cell cultures. Science 1959, 130(3373):432-437). However, since then, the fact that certain keto acids can be used as amino acid substitutes in high-performance cell culture and that they are suitable for overcoming the solubility and stability problems of some amino acids has received no attention at all.

Summary of the Invention

[0011] The present invention thus relates to a dry powder or dry granule cell culture medium directed to a liquid medium obtained after dissolution, wherein at least one alpha-keto acid of the group of 4-methyl-2-oxopentanoic acid (keto-Leu), 3-methyl-2-oxopentanoic acid (keto-Ile), alpha-ketoisovaleric acid (keto-Val), phenylpyruvic acid (keto-Phe) and alpha-ketogamma-methylthiobutyric acid (keto-Met), and / or derivatives thereof, is included in an amount such that the concentration of each keto acid and / or derivatives thereof is greater than 10 mM, preferably 20 - 600 mM, most preferably 30 - 300 mM. Typically, each keto acid is present at various concentrations, where typically 4-methyl-2-oxopentanoic acid (keto-Leu), 3-methyl-2-oxopentanoic acid (keto-Ile), alpha-ketoisovaleric acid (keto-Val), phenylpyruvic acid (keto-Phe) and / or derivatives thereof are present at higher concentrations greater than 50 mM, where alpha-ketogamma-methylthiobutyric acid (keto-Met) is typically present at lower concentrations, 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. This 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 preferred to have both amino acids and the corresponding keto acids and / or derivatives thereof in the medium formulation.

[0013] In another embodiment, the dry powder or dry granule cell culture medium contains two or more alpha-keto acids and / or derivatives thereof. In a preferred embodiment, the dry powder or dry granule cell culture medium contains the sodium salt of one or more of the alpha-keto acids listed above. In a preferred embodiment, the dry powder or dry granular 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 the sodium salts thereof.

[0014] The present invention is 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 derivatives thereof, preferably 4-methyl-2-oxopentanoic acid and / or 3-methyl-2-oxopentanoic acid and / or alpha-ketoisovaleric acid and / or derivatives thereof, wherein the resulting medium shows less color change and / or less precipitation when stored at 4°C or at room temperature for 90 days compared to a medium of the same composition but lacking the keto acid and / or derivatives thereof or having the keto acid and / or derivatives thereof replaced by the corresponding amino acid and / or derivatives thereof.

[0015] The present invention is further directed to a method for improving the solubility of a dry powder or dry granular 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-ketogamma-methylthiobutyric acid and / or derivatives thereof.

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

[0017] In a preferred embodiment, the method comprises providing a dry powder or dry granular cell culture medium in which the amino acids are substituted as described above, and dissolving said medium, which results in faster dissolution and / or occurs with less liquid compared to a medium of the same composition in other respects where the amino acids are not substituted. In another preferred embodiment, the dry powder or dry granular medium dissolves to give 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 granular cell culture medium with improved solubility comprises at least one saccharide component, 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 dry powder or dry granular cell culture medium with improved solubility dissolves to give a liquid medium containing solid components that dissolve in a solvent at 50 to 400 g / l, preferably 100 to 300 g / , and / or the concentration of each keto acid and / or its salt is greater than 10 mM, preferably 30 to 600 mM.

[0020] The present invention further provides a) mixing at least one alpha-keto acid of the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-ketogamma-methylthiobutyric acid and / or their derivatives with the other components of the cell culture medium b) subjecting the mixture from step a) to grinding towards a method for producing a dry powder cell culture medium according to the 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 relates to 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 are partially or wholly replaced by the corresponding keto acids selected from the group consisting of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-ketogamma-methylthiobutyric acid, and / or their derivatives c) incubating the mixture from step b) further towards a process for culturing cells.

[0023] In a preferred embodiment, the liquid cell culture medium contains each keto acid and / or its derivative present at a concentration of more than 10 mM. The present invention also relates to - filling a bioreactor with cells and an aqueous cell culture medium - incubating the cells in the bioreactor - adding continuously over the entire time of incubation of the cells in the bioreactor, or one or several times within said incubation time, a cell culture medium which is a feed medium, to the bioreactor Here, the feed medium has a pH of less than 8.5 and contains 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-ketogamma-methylthiobutyric acid and / or their derivatives, and 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 their salts at a concentration of 20 to 600 mmol / l, preferably 20 to 400 mmol / l each. 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 wholly replaced by the corresponding keto acids selected from the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid, alpha-ketogamma-methylthiobutyric acid, and / or their derivatives.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0037] The 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. The cell culture medium can contain all the components necessary to maintain and / or support the in vitro growth of cells, or it can contain only some components such that additional components are added individually. Examples of cell culture media according to the present invention are complete media that contain all the components necessary to maintain and / or support the in vitro growth of cells, as well as medium supplements or feeds. In a preferred embodiment, the cell culture medium is a complete medium, a perfusion medium or a feed medium. A complete medium is also called a basal medium and typically has a pH of 6.7 - 7.8. The feed medium preferably has a pH of less than 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. The feed or feed medium is not a basal medium that supports initial growth and production in cell culture, nor is it a cell culture medium added at a later stage to prevent nutrient depletion and maintain the production phase. Instead, it is a medium added at a later stage to sustain the production phase. The feed medium may have higher concentrations of some components compared to the basal culture medium. For example, some components such as nutrients including amino acids or carbohydrates may be present in the 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 medium is a mixture of components that maintain and / or support the in vitro growth of mammalian cells. Examples of mammalian cells are human or animal cells, preferably CHO cells, 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 contains no substances that are not chemically defined. This means that the chemical composition of all the chemicals used in the medium is known. Chemically defined media contain no yeast, animal, or plant tissues; 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 not known, exist with varying compositions, or can only be defined by extensive experimental efforts comparable to the evaluation of the chemical composition and structure of proteins such as insulin, albumin, or casein.

[0041] Powdery cell culture media or dry powder media are typically cell culture media resulting from a grinding process or a lyophilization process. It means that the powdery 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" 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 solvent, unless otherwise indicated.

[0042] Dry granular media are dry media resulting from a wet or dry granulation process. Preferably, it is a medium resulting from the roller compression of dry powder media. 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 solvent, unless otherwise indicated.

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

[0044] The size of the particles means the average diameter of the particles. The particle diameter is determined by the laser light scattering method (Mastersizer 3000, Malvern). The change in color of the liquid cell culture medium is preferably determined visually or spectroscopically. Precipitation can be determined visually or by nephelometry.

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

[0046] Various types of mills are known to those skilled in the art. A pin mill, also called a centrifugal impact mill, has protruding pins on a high-speed rotating disk that pulverize solids by providing breaking energy. 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, commercially available from Fitzpatrick (USA), uses a rotor with blades for grinding.

[0047] A process that is run continuously is a process that is not run in batch mode. If the process of grinding is run continuously, it means that the medium components are supplied to the mill permanently and steadily over a period of time. The cell culture medium according to the invention, in particular the complete medium, typically contains at least one or more sugar components, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffer components, one or more cofactors, and one or more nucleic acid components.

[0048] The medium may also contain surfactant components such as sodium pyruvate, insulin, vegetable protein, fatty acids and / or fatty acid derivatives and / or pluronic acid and / or chemically prepared nonionic surfactants. One example of a suitable nonionic surfactant is a difunctional block copolymer surfactant terminated with primary hydroxyl groups, also called a poloxamer (available, for example, 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 present invention are tyrosine, protein organic amino acids, especially 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 amino acids such as sodium salts, or their respective hydrates or hydrochlorides. For example, tyrosine means L- or D-tyrosine, preferably L-tyrosine, and its salts or hydrates or hydrochlorides.

[0050] Examples of vitamins are vitamin A (retinol, retinal, various retinoids and four carotenoids), vitamin B 1 (thiamine), vitamin B 2 (riboflavin), vitamin B 3 (niacin, niacinamide), vitamin B 5 (pantothenic acid), vitamin B 6 (pyridoxine, pyridoxamine, pyridoxal), vitamin B 7 (biotin), vitamin B 9(Folic acid, folinic acid), vitamin B12 (cyanocobalamin, hydroxocobalamin, 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 components 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 (CuSO 4 ·5H 2 O), sodium chloride (NaCl), calcium chloride (CaCl 2 ·2H 2 O), potassium chloride (KCl), iron(II) sulfate, ammonium iron citrate (FAC), sodium dihydrogen phosphate anhydrous (NaH 2 PO 4 ), magnesium sulfate anhydrous (MgSO 4 ), disodium hydrogen phosphate anhydrous (Na 2 HPO 4 ), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O), and zinc sulfate heptahydrate. Examples of buffers are CO 2 / HCO 3 (carbonate), phosphate, HEPES, PIPES, ACES, BES, TES, MOPS, and TRIS.

[0052] Examples of cofactors are thiamine derivatives, biotin, vitamin C, NAD / NADP, cobalamin, flavin mononucleotide and derivatives, glutathione, heme nucleotide phosphate and derivatives. According to the present invention, the nucleic acid components 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] The feed medium may have a different composition compared to the complete medium. They typically contain amino acids, trace elements, and vitamins. They may also contain saccharide components, although sometimes the saccharide components are added in a separate feed for production reasons.

[0054] A suitable feed medium may contain, for example, one or more of the following compounds: L - Asparagine monohydrate L - Isoleucine L - Phenylalanine Sodium 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 Sodium 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 (approx. 45% Se) Sodium dihydrogen phosphate monohydrate Ammonium iron(III) citrate (approx. 18% Fe).

[0056] According to the present invention, freezing means cooling to a temperature below 0°C. In the perfusion process, the cell culture medium is continuously added to the 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 possibility of reaching extremely high cell densities (due to regular medium exchanges), and the ability to remove the product from the bioreactor daily, thus reducing the exposure time to high temperatures, redox potential, or released cell proteases, and potentially enabling the production of extremely labile recombinant proteins.

[0057] The process for perfusion cell culture typically involves culturing cells in a bioreactor system including a bioreactor equipped with a medium inlet and a harvest outlet, where i. During the cell culture process, fresh cell culture medium is inserted into the bioreactor through the medium inlet continuously or one or several times, preferably continuously ii. During the cell culture process, the harvest is removed from the bioreactor through the harvest outlet continuously or one or several times, preferably continuously. The harvest typically contains the target 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 important building blocks of recombinant proteins produced using mammalian cell culture technology. The solubility of amino acids is a limiting factor that hinders the concentration of cell culture media and feed formulations. Such concentration is essential for developing next-generation manufacturing platforms. In particular, biomanufacturing processes that use in-line dilution reduce the volume of cell culture media that must be stored in tanks (= reduce the manufacturing footprint), or generally reduce the volume of feed added through fed-batch processes, thus increasing the volumetric titer. Therefore, highly concentrated formulations are required.

[0059] It has been found that some amino acids in cell culture media can be replaced with their 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. Apart from the solubility advantage, the use of keto acids has also been found to enable the reduction of ammonia in cell culture, which is a known toxic and inhibitory metabolite. Furthermore, the use of certain keto acids has been demonstrated to result in more stable formulations with less color change, no or delayed precipitation, and less or delayed formation of by-products when stored at RT.

[0060] Therefore, the keto acids and their salts of amino acids can be used in cell culture media formulations for the following applications. · Application 1: Increasing overall media / feed solubility · Application 2: Replacing the corresponding amino acid and reducing ammonium ion / ammonia formulations in cell culture · Application 3: To increase medium stability, reduce color change and precipitation caused by storage of the formulation 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

[0062] By partially or wholly substituting the amino acids leucine, isoleucine, valine, phenylalanine and / or methionine with the corresponding keto acids and / or their derivatives, the solubility of dry powder or dry granule cell culture media can be improved without having a negative effect on cell culture performance as compared to otherwise identical cell culture media. In a preferred embodiment, the sodium salts of the keto acids are used because 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, have high solubility in water compared to the corresponding amino acids, and they can replace the corresponding amino acids in their role of either cooperating intracellularly to return to the corresponding amino acid or otherwise maintaining and / or supporting the in vitro growth of cells. 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] The peptide derivative is a derivative in which one or more, typically one, two or three amino acids are linked to a keto acid via a peptide bond. In the case of keto-leucine, the structural formula of the peptide derivative is shown in Scheme 1 below: [Chemical formula] R 1 is an 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 the 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: [Chemical formula] R 2 is alkyl or aryl, and the alkyl group may be further substituted with -OH or OR 2 and may also form, for example, an ether or an ester.

[0066] Examples of suitable R 2 are methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, benzyl and [Chemical formula] etc.

[0067] Other derivatives are shown in Scheme 3: [Chemical formula]

[0068] The above example shown for ketoleucine can of course be equally realized for other keto acids of the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-ketogamma-methylthiobutyric acid.

[0069] The present invention is thus directed to a dry powder or dry granule cell culture medium comprising at least one alpha-keto acid of the group of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-ketogamma-methylthiobutyric acid and / or derivatives thereof, 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 acid 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 more than 10 mM, preferably 20 - 600 mM, most preferably 30 - 300 mM.

[0071] In one embodiment, the dry powder or dry granule cell culture medium containing the 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 the keto acid as defined above contains up to 50% (mol%) of the corresponding amino acid. For use of the dry powder or dry granule medium, a solvent, preferably water (most specifically distilled water and / or deionized water, or purified water or water for injection) or an aqueous buffer is added to the medium and the components are mixed until the medium is completely dissolved in the solvent.

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

[0073] Dry powder or dry granule culture medium compositions containing higher concentrations of leucine, isoleucine, valine, phenylalanine, and methionine show turbidity when mixed with a solvent due to the limited solubility of the amino acids, while the cell culture medium according to the present invention using the corresponding keto acids and / or their derivatives at the same concentration gives a clear solution. This is particularly suitable for feed media.

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

[0075] In some cases, it may be advantageous to modify the amount of keto acid, particularly increase it, compared to the amount of amino acid being replaced. For the replacement of isoleucine, leucine, and valine, typically a 1:1 replacement is sufficient, while for phenylalanine and methionine, it has been found that it is preferably to add more keto acid compared to the amount of amino acid. Typically, a replacement of 1:1.1 to 1:3 (molar basis) is suitable.

[0076] Preferably, by completely substituting the amino acids leucine, isoleucine, valine, phenylalanine, and methionine with the corresponding keto acids and / or their derivatives, particularly the sodium salts of the keto acids, the maximum solubility of the medium can be increased. As can be seen in Example 3, the solubility of the dry powder medium can be doubled, for example. In addition to the improvement in the solubility of the dry powder or dry granule medium by substituting the amino acids as described above, it was unexpectedly found that when a medium in which leucine and / or isoleucine is replaced with the corresponding keto acid and / or its salt is used, the specific productivity of cell culture increases.

[0077] Three important quality characteristics of IgG1 produced using a medium in which leucine and / or isoleucine is replaced with the corresponding keto acid and / or its salt could further show that there is no difference between the control conditions using amino acids and the substituted conditions. The three important quality characteristics are glycosylation pattern, antibody aggregation and fragmentation, and charge variants.

[0078] It was further found that keto acids and / or their derivatives, particularly the 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. The liquid medium containing the above components does not contain 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, and / or their salts, but shows less color change when stored at either room temperature or 4°C for 3 months, regardless of the presence or absence of light exposure, compared to a medium containing the same amount of the corresponding amino acids. These also showed a decrease in precipitation.

[0079] This effect can be achieved when the corresponding amino acids, such as isoleucine and / or leucine, are replaced with the corresponding keto acids and / or their derivatives. This can also be achieved when the corresponding keto acids, such as 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid and / or their derivatives, are added to a cell culture medium preparation containing leucine and / or isoleucine. This means that keto acids and / or their derivatives can be used as a medium stabilizer regardless of the medium composition. A suitable concentration in the liquid preparation 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 the components and grinding them. Mixing the components is well known to those skilled in the art for producing a dry powdered cell culture medium by grinding. Preferably, all the components are thoroughly mixed so that all parts of the mixture have almost the same composition. Regarding homogeneous cell growth, the higher the uniformity of the composition, the better the quality of the resulting medium. Grinding can be carried out by any type of mill suitable for the production of cell culture media. Typical examples are ball mills, pin mills, Fitz mills, or jet mills. Preferably, a pin mill, Fitz mill, or jet mill, most preferably a pin mill. Those skilled in the art know how to operate such mills.

[0081] A large-scale device mill with a disk diameter of about 40 cm is typically run at 1 - 6500 revolutions per minute, preferably 1 - 3000 revolutions per minute in the case of a pin mill, for example. Grinding can be carried out under standard grinding conditions, resulting in a powder having a particle size between 10 and 300 μm, most preferably between 25 and 120 μm.

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

[0083] Preferably, all components of the mixture to be comminuted are dry. This means that if they contain water, they contain only water crystals that are not more than 10%, preferably not more than 5%, and most preferably not more than 2% by weight of unbound or non - aggregated water molecules. In a preferred embodiment, the comminution is carried out in an inert atmosphere. The preferred inert protective gas is nitrogen.

[0084] In a further preferred embodiment, all components of the mixture are frozen prior to comminution. Freezing of the components prior to comminution 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, the freezing is done by liquid nitrogen. This means that, for example, prior to introduction into the mill, liquid nitrogen is poured into the container in which the components are stored, so that the components are treated with liquid nitrogen. In a preferred embodiment, the container is a feeder. When the container is a feeder, preferably the liquid nitrogen is introduced next to or near the feeder into which the components are fed.

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

[0086] In a preferred embodiment, all components are placed in a container into which the mixture is transferred, preferably into a feeder, most preferably a metering screw feeder. In the feeder, the components are sometimes further mixed - depending on the type of feeder - and additionally cooled. The frozen mixture is transferred from the feeder to the mill such that the mixture to be ground 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 components in the feeder) is more than 1 minute, preferably between 15 and 60 minutes. The metering screw feeder (also called a lightweight snail) is typically run at a speed of 10 to 200 revolutions per minute, preferably 40 to 60 revolutions per minute.

[0088] Typically, the temperature for grinding is maintained between -50 and +30 °C. In a preferred embodiment, the temperature is maintained at approximately 10 °C. The oxygen level during grinding 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 invention is carried out continuously by permanently filling the feeder with the mixture of components for cooling over a period of time and permanently filling the mill with the cooled mixture from the feeder.

[0089] The present invention relates to a) providing a bioreactor b) providing a liquid cell culture medium containing 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-ketogamma-methylthiobutyric acid and / or their derivatives, preferably at a concentration above 10 mM c) mixing the cells to be cultured with the liquid cell culture medium d) incubating the mixture of step b) It is further directed to a process for culturing cells according to

[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 replaced by the corresponding keto acids selected from the group consisting of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-ketogamma-methylthiobutyric acid and / or their derivatives.

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

[0092] The present invention has been found to be extremely suitable for the preparation of feed media. Due to the limitation of the availability of certain amino acids required in the feed medium, the concentration of the feed medium is limited due to solubility problems.

[0093] As a result, a feed medium is required that contains all the required components at a high concentration in one feed. In addition, the pH of the feed should not have a negative impact on cell culture, i.e., the pH of the liquid feed should be less than 8.5, preferably between 6.5 and 7.8.

[0094] It has been found that by partially or preferably completely substituting the amino acids isoleucine, leucine, valine, phenylalanine and methionine with the corresponding keto acids and / or derivatives, preferably their salts, the solubility of the resulting dry powder medium is improved. This suggests the possibility of producing liquid media containing higher concentrations of components, so that the same amount of components can be added to cell cultures in a smaller volume of liquid, and yet, preferably, at a suitable pH of less than 8.5. A more highly concentrated feed medium containing keto acids can be used without any negative effects on cell growth and / or productivity and the stability of the liquid medium, and in some cases even with positive effects.

[0095] Thus, the present invention also relates to any feed medium 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 consisting of 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, alpha-ketoisovaleric acid, phenylpyruvic acid and alpha-ketogamma-methylthiobutyric acid and / or derivatives thereof, 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 - 8.4.

[0096] The present invention also relates to - filling a bioreactor with cells and an aqueous cell culture medium - incubating the cells in the bioreactor - continuously, or one or more times during the incubation time, adding to the bioreactor a cell culture medium which is a feed medium over the entire time of incubation of the cells in the bioreactor Here, the feed medium preferably has a pH of less than 8.5 and contains 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-ketogamma-methylthiobutyric acid and / or their derivatives. It is directed to a fed-batch process for culturing cells in a bioreactor according to .

[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 to 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 salt. Typically, the feed medium contains 50 to 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 added continuously to the bioreactor during incubation or one or several times within 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 into this specification.

[0099] Example The following examples represent 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 to the solubility of their respective keto acids or their salts in water at 25°C through the preparation of saturated solutions. After sedimentation, the solution was dried using infrared rays (120°C, 120 minutes), and the residual mass was determined in g / kg. As shown in Figure 1, the solubility of keto acids and their salts was significantly higher compared to the solubility of the respective amino acids in water. To rule out that the increase in solubility was due to the sodium salt form of the keto acids, another experiment was conducted to compare the solubility of Leu, sodium Leu salt, and keto Leu sodium salt. The maximum solubility obtained in water was 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 important, thus showing that it was not due only to the salt form.

[0100] Example 2: Maximum solubility of keto acids compared to their respective amino acids in 4Feed lacking Ile and Leu Increased amounts of keto acids and their salts were added to a cell culture feed formulation lacking Ile and Leu (Cellvento® 4Feed, MilliporeSigma). Similarly, as a control, increased 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 an amino acid or a keto acid, the feed was stirred for 10 minutes and the turbidity was measured. The experiments were conducted at room temperature (25 °C).

[0101] The maximum solubility of Ile in Cellvento® 4Feed lacking Ile / Leu was approximately 105 mM, while it was found that keto Ile was soluble at turbidity values below 5 NTU even at the maximum test concentration of 635 mM tested (see Figure 1). This shows that in 4Feed lacking Ile / Leu, keto Ile is at least more than 6-fold more soluble than Ile. The maximum solubility of Leu in Cellvento® 4Feed lacking Ile and Leu was approximately 90 mM, while for keto-Leu, the maximum solubility concentration (turbidity value less than 5 NTU) was found to be 240 mM (see Figure 2). This indicates that in 4Feed lacking Ile / Leu, keto-Leu is more than 2.6 times more soluble than Leu.

[0102] Example 3: The use of keto acids enables the concentration of cell culture medium formulations at neutral pH. The maximum solubility of Cellvento® 4Feed was determined by dissolving increasing amounts of the 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. Osmotic pressure and turbidity were measured (see Figure 3). The data indicate that a 1.2-fold concentrate of this formulation is already insoluble, as particles were detected in the suspension and the turbidity far exceeded the limit of 5 NTU.

[0103] Since Ile and Leu were identified as the first limiting amino acids for the concentration of the Cellvento® 4Feed formulation, a new backbone feed lacking Ile and Leu was produced (4Feed-Ile / Leu). The maximum concentration of this feed was determined by dissolving increasing amounts of the feed dry powder medium in water until precipitation was visually detected, with or without the supplementation of keto-Leu and keto-Ile. 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 indicate that the maximum solubility of Ile / Leu-deficient Cellvento® 4Feed was approximately 228 g / L. When keto-Leu and keto-Ile were added, the maximum solubility was obtained at 216 g / L to 228 g / L of the deficient dry powder medium supplemented with a combined amount of 36 g / L to 38 g / L of keto-Leu and keto-Ile (molar ratio corresponding to the theoretical amount of Ile and Leu in the concentrate), resulting in a total concentration of 252 g / L to 266 g / L of the formulation containing both keto-Leu and keto-Ile. Considering that Cellvento® 4Feed has a concentration of 130 g / L, replacing Ile and Leu with keto-Ile and keto-Leu represents a 100% increase in concentration. The data shows that it is possible to concentrate the formulation up to at least 2x (265 g / L) as no particles could be detected in the suspension and the turbidity was less than 5 NTU (see Figure 4).

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

[0106] As shown in Figure 5A, the feeds containing Ile and Leu under control conditions 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% under light protection and light exposure conditions, respectively. At RT, the decrease was even more significant, and in the keto-Leu condition, the AUC decreased by 31% (light protection) and 37% (light exposure), respectively. This indicates that replacement of Leu with keto-Leu can significantly reduce the color change observed over time in the feed.

[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, the AUC decreased by 33% and 68% under light protection and light exposure conditions, respectively. At RT, no decrease was seen under light protection conditions, but a 38% decrease was observed under light exposure conditions, indicating that replacement of Ile with keto-Ile can significantly reduce the color change observed over time in the feed. Our overall results suggest 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, regardless of the presence or absence of light exposure.

[0108] In addition, when keto-Leu was used instead of Leu in the feed, precipitation of the feed was delayed. To observe the precipitation, 50 mL Falcon tubes were inverted to check for potential precipitation at the bottom of the tubes and photographed. No precipitation occurred at 4 °C under any conditions, but at RT with light protection, precipitation occurred between D49 and D70 under the control conditions, while no precipitation was observed under the conditions containing keto-Leu. Complete inhibition of precipitation was not observed in RT upon exposure to light, but precipitation was delayed under the keto-Leu conditions. Precipitation was observed from D49 under the control conditions, while the first precipitation appeared at D70 under the keto-Leu conditions. Over the next few days, under the conditions containing keto-Leu, the amount of precipitate and the intensity of the precipitate color decreased, indicating that the stability of the keto-Leu formulation increased slightly even under RT light exposure.

[0109] Finally, the amount of ammonium ions formed during storage of the feed containing keto acids at 4 °C or RT was less when compared to the feed containing normal amino acids. To be able to evaluate the formation of NH 3 over the entire duration of the stability test, the AUC of the NH 3 concentration was calculated for a 3-month time frame and the conditions were compared. The results for keto-Leu are presented in Figure 6A, showing less ammonia formation when compared to the control conditions. When the feed was stored at 4 °C with light protection and light exposure, ammonia was produced 10% and 19% less, respectively, under the keto-Leu conditions compared to the control. At RT, the same trend was observed, and when the feed was stored for 3 months with light protection and light exposure, ammonia levels decreased by 15 and 5%, respectively.

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

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

[0112] The viable cell density (VCD) and viability were evaluated using a Vi-CELL XR (Beckman Coulter, Fullerton, CA). The metabolite concentration was monitored using a Cedex Bio HT (Roche Diagnostics, Mannheim, Germany) based on spectrophotometry and turbidimetry. The quantification of amino acids was performed by UPLC after derivatization using an AccQ·TagUltra® reagent kit. Derivatization, chromatography, and data analysis were carried out according to the supplier's (Waters, Milford, MA) recommendations.

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

[0114] NH 3 is an undesirable metabolite produced over the course of the fed-batch process. The amount of NH 3 produced during the 17-day fed-batch process under the keto-Leu and keto-Ile conditions (Figure 9A) was significantly decreased compared to the control containing Leu and Ile, indicating that either a significant portion of ammonia is generated from the oxidative deamination of Leu and Ile or the presence of keto acids in the bioreactor medium promotes the utilization of free NH 3 as a building block for generating amino acids by amination.

[0115] The concentration of amino acids in the spent medium was determined. Under the condition where Leu was replaced by 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), but 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. Under the condition where Ile was replaced by keto - Ile (Figure 10A), the concentration of Ile detected over time was significantly lower than the Ile concentration in the positive control, indicating either that the conversion from keto - Ile to Ile is slow or that another product is being formed from keto - Ile during the culture. Comparison of the keto - Ile condition with the negative control where the feed was deficient in Ile and Leu nonetheless showed that Ile can be produced from keto - Ile in that fed - batch. Additionally, careful analysis of the chromatogram enabled the identification of a new peak corresponding to allo - Ile that increased over time (Figure 10B).

[0116] The quality of the antibody produced in the control fed - batch process (feed containing Ile and Leu) was compared with the quality of the antibody produced in feeds lacking either Leu or 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, the glycosylation patterns were analyzed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF). Briefly, the purified antibodies were denatured and immobilized, digested with N-Glycanase®, and the glycans were released from the antibodies by labeling with APTS at 50 °C for 60 minutes. After a washing 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 minutes. The inlet and outlet buffer vials were changed every 20 cycles. Samples were introduced by pressure injection at 0.5 psi for 12 seconds, followed by a 0.2-minute immersion step to wash the capillary tip. Finally, separation was performed at 20 kV for 20 minutes with reverse polarity applied for 0.17 minutes. Peaks were identified according to their individual migration times and integrated according to the following parameters: peak width 0.05, threshold 10,000, shoulder sensitivity 9,999.

[0118] Antibody aggregation and fragmentation were measured using size exclusion chromatography on a Waters 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, and the flow rate was 0.35 mL / min. The sample concentration was adjusted to 1.0 mg / mL using the storage buffer after IgG purification, and detection was performed using absorbance at 214 nm.

[0119] Charge variants were measured on a Capillary Electrophoresis CESI 8000 (Beckman Coulter / Sciex) using cIEF according to the manufacturer's instructions. The sample concentration was adjusted to a concentration of 1.5 mg / mL using the storage buffer after IgG purification. Prior to measurement, the sample was mixed with a master mix containing various pH markers, cathode / anode stabilizers, 3 M urea cIEF gel, and Pharmalyte. The results obtained for glycosylation (Figure 11), high molecular weight species and low molecular weight species (Figure 12A), and charge variants (Figure 12B) showed no difference between the control conditions and the conditions where Ile and Leu were replaced with keto-Ile and keto-Leu, indicating that the amino acid exchange did not affect three important quality attributes of the IgG1 produced in this study.

[0120] Example 6: Confirmation of keto-Leu performance using CHODG44 and CHOK1 clones producing IgG1. The applicability of the technology of the present invention to various bioprocesses was demonstrated by performing fed-batch experiments using other types of CHO cells: CHODG44 and CHOK1 (non-GS) for keto-Leu as an example. The results of the DG44 cell line (Figure 13) show that the VCD is lower and the IgG titer is also slightly lower under the keto-Leu condition compared to the control. Nevertheless, in the process using keto-Leu, the overall specific productivity increased slightly. The spent medium data indicate that for this cell line, the Leu concentration under the keto-Leu condition is also almost the same as the control Leu concentration, confirming that Leu can be produced very rapidly from keto-Leu in this cell line. Figure 13: Performance of the keto-Leu-containing process of the CHODG44 cell line expressing IgG1 compared to the control. Figure 14: Performance of the keto-Leu-containing process of the CHOK1 non-GS cell line expressing IgG1 compared to the control.

[0121] Example 7: Performance in batch using various seeding densities and various leu / keto-Leu ratios The results of our spent fed-batch media obtained with three different CHO cell lines using keto-Ile and keto-Leu indicate that the formation of Ile, allo-Ile, and Leu from the keto acids is very rapid. This indicates that since the keto acids are readily available from the start of the culture, they may also be used to increase the solubility of batch and perfusion media. To confirm that this is applicable in the CHO system, keto-Leu or keto-Ile was used in the cell culture medium (Cellvento® 4CHO) as an alternative to Leu and Ile, respectively. The Leu / Ile-deficient version of the formulation was produced, and equimolar concentrations of keto-Leu vs. Leu and keto-Ile vs. Ile were used (Figure 15). The cell growth and viability of the CHOK1GS cell line were monitored in serial passage experiments over several weeks to confirm that growth was not due to the remaining 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. Also, the quantification of amino acids in the spent media was used to track amino acid production over time.

[0122] In a similar manner, batch experiments at higher cell seeding densities were performed in media containing various ratios of Leu / keto-Leu to understand which ratio is preferred when starting at higher cell densities (Figure 16). The analytical methods used were the same as those described above. The results of the serial passages indicate that the CHOK1GS cells cannot grow in media deficient in Ile and Leu because no growth was observed during the first few days of culture and the viability decreased significantly. In contrast, continuous growth was observed when Leu or Ile was replaced by their corresponding keto acids. Overall, the maximum viable cell density observed in each passage was slightly lower than the control condition containing Ile and Leu, indicating that a small amount of Leu and Ile is required to obtain performance equivalent to the control condition. This amount can be very easily determined experimentally by testing media containing various ratios of Ile / Leu and keto-Ile / keto-Leu.

[0123] In batch experiments, performance was equivalent between control conditions and those with keto-Leu, indicating that CHOK1GS cells can grow when Leu is replaced with a molar equivalent of keto-Leu (Figure 15A). Similar amounts of IgG were detected on days 7 and 10 under those conditions (Figure 15B). In contrast, when Ile was replaced with keto-Ile, growth and IgG concentration after 5 days were slightly impaired, indicating that a small amount of Ile may be required to obtain growth and titers similar to the control under batch conditions over time. This amount can be experimentally determined very easily by testing media containing various ratios of Ile and keto-Ile. Alternatively, higher molar concentrations of keto-Ile may be tested relative to the Ile concentration.

[0124] The performance difference between keto-Ile and keto-Leu may be explained by looking at the formation of Ile, allo-Ile, and Leu in the spent media. While 34% of the initial keto-Leu concentration was detected as 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 as allo-Ile by day 10. This indicates that the amination of keto-Leu to Leu by the cells is more efficient than the amination of keto-Ile to Ile due to the accompanying formation of allo-Ile, which may not be utilized by the cells to the same extent as Ile.

[0125] Finally, batch experiments were performed at higher cell densities 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 under such conditions. In this experiment, the CHOK1GS cell line was seeded at 0.3, 0.6, or 1.10^6 cells / mL into media containing 0, 25, 50, 75, or 100% keto-Leu with the remainder added in the form of leucine.

[0126] As expected, the results show an increase in growth and titer with increasing seeding density. Among the various ratios of keto Leu / Leu, the maximum VCD was observed at the highest seeding density of 1.10^6 cells / mL with 100% keto Leu replacement. 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 ratio of keto Leu:Leu (keto Leu 50%, Leu 50%). For the titer, no significant difference was seen for seeding at 0.3 and 1.10^6 cells / mL, while a slight tendency was observed for seeding at 0.6.10^6 cells / mL that the IgG concentration increased with a higher ratio of Leu / keto Leu. This difference may not be significant.

[0127] Example 8. Performance of other keto acids against their respective amino acids in FB culture In FB experiments, other keto acids were tested as replacements for their respective amino acids. When Val in the feed was replaced with keto Val, behavior similar to that of Ile and Leu was observed (Figure 17). Indeed, similar VCD and titers were observed compared to the positive control, while the feed lacking Val led to a significantly decreased VCD and a very low titer after 7 days. NH 3 Concentration also decreased when keto acids were used, and for Val, the utilization of each keto acid also led to less NH during fed-batch culture. 3 Overall, this indicates that keto Val, like members of the branched-chain keto acids, is likely to exhibit the same behavior as keto Leu and keto Ile and is highly likely to be aminated very rapidly during cell culture. Due to the structural similarity with keto Ile and keto Leu, the effect of keto Val on the overall feed concentration and feed stability is similar to that of other branched-chain keto acids. Six times higher solubility in water was confirmed compared to Val.

[0128] Regarding phenylalanine (Phe) and its keto acid, phenylpyruvic acid (Figure 18), the amination reaction that produces Phe in cell culture appears to be slower than the amination reaction that occurs with branched-chain keto acids. In fact, when Phe was replaced with equimolar concentrations of phenylpyruvic acid, the spent media data showed 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 the control conditions. After 5 days, a significantly decreased VCD was observed, and a final decrease in titer of 20% was observed. Following this result, conditions where 2x molar equivalents of Phe were used as the concentration of phenylpyruvic acid in the feed. The results indicate that an increase in the amount of phenylpyruvic acid was able to recover the VCD, titer, and a similar amount of Phe in the spent media. These data confirm that Phe can also be replaced with its keto acid, but concentration adjustment may be necessary to account for the slow pace of the amination reaction.

Claims

1. - Filling a bioreactor with cells and an aqueous cell culture medium - Incubating the cells in the bioreactor - Continuously throughout the total time of incubation of the cells in the bioreactor, or one or more times within said incubation time, adding to the bioreactor a cell culture medium which is a liquid feed medium A fed-batch process for culturing cells in a bioreactor, comprising: wherein the feed medium has a pH of less than 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, and / or their salts, and the cells are CHO cells, said fed-batch process.

2. The fed-batch process according to claim 1, wherein the feed medium comprises at least 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, and / or their salts at a concentration of 12 to 600 mmol / l.

3. The fed-batch process according to claim 1, wherein the feed medium comprises 50 to 400 g / l of solid components dissolved in a solvent.

4. The fed-batch process according to claim 1, wherein the feed medium added to the bioreactor continuously during incubation, or one or more times within said time, always has the same composition.

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