Cell culture medium

Abstract

The present invention relates to a cell culture medium comprising one or more N-lactoyl derivatives of amino acids. The poor solubility of some amino acids in cell culture media is overcome by substituting them with N-lactoyl derivatives.

Description

Technical Field

[0001] The present invention relates to a cell culture medium containing N-lactyl derivatives of one or more amino acids. The poor solubility of some amino acids in cell culture media is overcome by partially or completely replacing them with N-lactyl derivatives.

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 supporting 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, a major advance was 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 growing 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 media are 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 product (antibody or recombinant protein) in the bioreactor, the feed medium is highly concentrated. The controlled addition of nutrients directly affects the growth rate and lifespan of the culture.

[0008] Amino acids (AAs) are essential components of cell culture media as they are important for supporting cell growth. In addition, AAs are important building blocks of recombinant proteins produced using mammalian cell culture technology. The solubility of AAs is a limiting factor that hinders the concentration of cell culture media (CCM) and feed formulations. Such concentration is essential for the development of next-generation manufacturing platforms. In particular, highly concentrated formulations are required for biomanufacturing processes that use in-line dilution to reduce the volume of CCM that has to be stored in tanks (= reduce the manufacturing footprint), or generally to reduce the volume of feed added throughout the fed-batch (FB) process and thus increase the potential volumetric titer. As a result, it is desirable to find ways to improve the solubility of amino acids.

[0009] N-lactyl amino acids are known to have high resistance to proteolysis (Lorbert SJ et al: Oligomers and oligomeric segments of alpha-hydroxy carboxylic acids and alpha amino acids. US 2004 / 0048347, 2004), but it has unexpectedly been found that the amino acids in cell culture media can be replaced with their N-lactyl derivatives or their salts. In addition to their use as amino acid sources, the N-lactyl derivatives exhibit high solubility compared to their corresponding amino acids and can therefore be used in highly concentrated formulations.

[0010] N-lactyl amino acids are known in the food industry, particularly in foods such as soy sauce and meat products, as taste-active amino acids formed from free amino acids by the action of lactoyltransferase of the lactobacillus species (Zhao CJ, Schieber A, Ganzle MG: Formation of taste-active amino acids, amino acid derivatives and peptides in food fermentations - A review. Food Res Int 2016, 89(Pt 1):39-47).

[0011] N-lactyl-amino acids have also been described in the context of medicine or human cell models. According to the study by Jansen RS et al., N-lactyl-amino acids are ubiquitous metabolites derived from the CNDP2-mediated reverse proteolysis of lactate and amino acids (Proc Natl Acad Sci U S A 2015, 112(21):6601-6606). In a very recent study, N-lactyl-amino acids have been described as extracellular biomarkers that correlate with intracellular amino acid concentrations in human cell models. This derivative has been described as a useful biomarker for distinguishing tumor cells from normal cells: Knott ME, Manzi M, Zabalegui N, Salazar MO, Puricelli LI, Monge ME: Metabolic Footprinting of a Clear Cell Renal Cell Carcinoma in Vitro Model for Human Kidney Cancer Detection. J Proteome Res 2018, 17(11):3877-3888. N-lactyl-amino acids as a cell culture medium component are not known.

Summary of the Invention

[0012] The present invention is thus directed to a cell culture medium comprising at least one N-lactyl-amino acid. In the following, when the term amino acid is used, it means free amino acids and their salts such as Na + , K + , Mg 2+ , Ca 2+ , Li + , preferably their Na + salts. Those skilled in the art will recognize that free amino acids can be used or that H + can be replaced by metal counterions such as Na + to form salts.

[0013] In a preferred embodiment, the N-lactyl-amino acid is selected from N-lactyl-leucine, N-lactyl-isoleucine, N-lactyl-valine, N-lactyl-phenylalanine, N-lactyl-tyrosine and / or N-lactyl-methionine, most preferably N-lactyl-leucine and / or N-lactyl-isoleucine.

[0014] In a preferred embodiment, the cell culture medium comprises one or more of Formula I:

Chemical formula

Chemical formula

[0015] When the amino acid is isoleucine, the components of Formula I are

Chemical formula

[0016] In a preferred embodiment, the cell culture medium comprises the sodium salt of N-lactyl amino acid. It preferably means that R 1 + is Na + . In a preferred embodiment, the cell culture medium is a dry powder medium.

[0017] In one embodiment, particularly when the cell culture medium is a basal medium or a perfusion cell culture medium, it contains one or more N-lactyl amino acids and the corresponding native amino acids. This means that it contains, for example, N-lactyl-leucine and the corresponding native leucine. In this case, native means an unmodified amino acid and / or its salt. In another preferred embodiment, the cell culture medium is a feed medium.

[0018] The feed medium may contain one or more N-lactyl amino acids and the corresponding native amino acids, but it may also contain only one or more N-lactyl amino acids and not contain the corresponding native amino acids. In a preferred embodiment, when the cell culture medium is a feed medium, the medium contains one or more N-lactyl amino acids but does not contain the corresponding native amino acids.

[0019] In another preferred embodiment, the cell culture medium is a liquid medium having a pH of 8.5 or less and containing at least one N-lactyl amino acid of formula I at a concentration of more than 10 mmol / l. In the case of a feed medium, the concentration is typically more than 30 mmol / l. The upper limit is defined only by the solubility of the lactyl amino acid. Solubility can depend on the solvent, pH and salt concentration. As a result, it is typically possible to produce a liquid medium within a concentration of lactyl amino acid up to 500 mmol / l or more.

[0020] In a preferred embodiment, the pH of the liquid medium is from 6.0 to 8.5, most preferably from 6.5 to 7.8. In one embodiment, the cell culture medium contains 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.

[0021] The present invention further a) Mixing one or more N-lactyl-amino acids according to formula I with the other components of the cell culture medium b) Subjecting the mixture from step a) to milling directed to a method for producing a cell culture medium according to the invention. 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 milling.

[0022] The invention further relates to a) Providing a bioreactor b) Mixing the cells to be cultured with a cell culture medium according to the invention c) Incubating the mixture from step b) directed to a process for culturing cells. In one embodiment, the bioreactor is a perfusion bioreactor.

[0023] The invention also relates to - Filling a bioreactor with cells and an aqueous cell culture medium - Incubating the cells in the bioreactor - Continuously, or once or several times over the entire time during the cell incubation time, adding to the bioreactor a cell culture medium which is a feed medium in this case wherein the feed medium is a cell culture medium according to the invention containing at least one N-lactyl amino acid directed to a fed-batch process for culturing cells in a bioreactor. Preferably, the feed medium has a pH of less than 8.5 and contains at least one N-lactyl amino acid at a concentration of more than 10 mmol / l. Preferably, the N-lactyl amino acid is N-lactyl leucine and / or N-lactyl isoleucine.

Brief Description of the Drawings

[0024]

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Modes for Carrying Out the Invention

[0025] An N-lactyl amino acid is an amino acid covalently linked via its amino group to a lactyl residue. The lactyl residue is the chemical moiety CH 3 CH(OH)CO- of lactic acid. The N-lactyl amino acids according to the present invention are, by way of example, products that can be obtained by chemical or biological synthesis. N-lactoyl amino acids can be synthesized in vivo and in microorganisms by enzymes such as lactoyl transferase of the Lactobacillus species (Zhao CJ, Schieber A, Ganzle MG: Formation of taste-active amino acids, amino acid derivatives and peptides in food fermentations - A review. Food Res Int 2016, 89(Pt 1):39-47) or by CNDP2-mediated reverse proteolysis (Jansen RS, et al: N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids. Proc Natl Acad Sci U S A 2015, 112(21):6601-6606).

[0026] The synthesis of N-lactoyl amino acids using a biocatalytic approach has also been described in mixtures containing an enzyme (an enzyme that catalyzes the formation of peptide bonds such as, for example, serine protease, thiol protease, metalloprotease, esterase or alkaline protease), an alpha-hydroxycarboxylic acid (or a derivative such as the corresponding ester, acid halide, amide, anhydride or ketene) and an alpha amino acid (or a derivative such as the corresponding ester, acid halide, amide, anhydride or ketene) (Lorbert SJ et al: Oligomers and oligomeric segments of alpha-hydroxy carboxylic acids and alpha amino acids. US 2004 / 0048347, 2004).

[0027] Several chemical processes have been described for the synthesis of N-lactyl amino acids. The synthesis of Lac-Phe from Sforza S, Cavatorta V, Galaverna G, Dossena A, Marchelli R: Accumulation of non-proteolytic aminoacyl derivatives in Parmigiano-Reggiano cheese during ripening. International Dairy Journal 2009, 19(10):582-587 is illustratively described in Example 1. Other synthetic processes have been described in the literature. For example, in Frerot E, Chen T: Identification and quantitation of new glutamic acid derivatives in soy sauce by UPLC / MS / MS. Chem Biodivers 2013, 10(10), Lac-Glu was synthesized from L-lactic acid and L-glutamic acid ester, mainly producing L-type Lac-AA. Characterization was performed using LC-MS SRM. Similarly, Lac-Val was synthesized in two steps from L-lactic acid and H-Val-OBzl tosylate, and the overall yield after hydrogenolysis was 56%. Finally, the synthesis by a single coupling reaction between amino ester-protected amino acids and (+)-(S)-lactic acid in the presence of 1 equivalent of PyBOP® (benzotriazolyl-oxy-tris[pyrrolidino]-phosphonium hexafluorophosphate) and excess diisopropylethylamine is described in Frerot E, Escher D: Flavored products and a process for their preparation. US5780090, 1998. NMR and LC-MS data were provided for Lac-Glu, Lac-Ala, Lac-Leu, Lac-Ile, N-lactyl-tyrosine (Lac-Tyr) and Lac-Met.

[0028] The N-lactyl amino acid is preferably a compound of formula I [Chemical formula] In the formula, R 1 + is H + or Na + , K + , Mg 2+ , Ca 2+ , Li + such as metal ions, preferably Na + and R 2 is a characteristic residue of an amino acid .

[0029] The characteristic residue of an amino acid is the unique part of the amino acid. The characteristic residue is the part that gives the amino acid characteristic properties so that the amino acid can be distinguished from other amino acids. For example, some characteristic amino acid residues are polar, others are non-polar, some characteristic amino acid residues are aliphatic, and others are aromatic. Figure 1 shows the general structure of an amino acid having some exemplary characteristic residues R 2 .

[0030] 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 the cell culture medium 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 or a feed medium. A complete medium, also called a basal medium, typically has a pH of 6.5 to 7.8. A feed medium preferably has a pH of less than 8.5, preferably 6.0 to 8.5.

[0031] 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 the basal medium that supports the initial growth and production in cell culture, nor is it the cell culture medium added at a later stage to prevent nutrient depletion and maintain the production phase, but rather the 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.

[0032] A 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.

[0033] 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 the chemicals used in the medium is known. Chemically defined media do not contain any yeast, animal, or plant tissues; they do not contain feeder cells, serum, extracts, or digests, or any other components that may contribute to proteins that are chemically incompletely defined in the medium. 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 albumin or casein.

[0034] Powdery cell culture media or dry powder media are typically cell culture media resulting from a milling process, a lyophilization process, or a dry or wet granulation 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. Dry powder media resulting from a milling or lyophilization process typically have a particle size of less than 0.5 mm, for example, 0.05 - 0.5 mm.

[0035] Dry powder media obtained from dry or wet granulation processes, for example by spray drying, wet granulation, or dry compression, typically have a particle size greater than 0.5 mm, for example 0.5 - 5 mm. Dry compression is typically performed on a roll press. US 6,383,810 B2 discloses a method for producing aggregated eukaryotic cell culture media powder. This method includes wetting a dry powder cell culture media with a solvent and then redrying the wetted media to obtain a dry aggregated cell culture media. In one aspect, the dry powder media according to the present invention are produced by dry compression.

[0036] 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.

[0037] The size of the particles means the average diameter of the particles. When the size of the particles is given, it means that at least 80%, preferably at least 90% of the particles have the given particle size or are within the given particle size range. The particle diameter is determined by laser light scattering method.

[0038] 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.

[0039] 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 provide breaking energy to pulverize solids thereby. For example, pin mills are sold by Munson Machinery (USA), Premium Pulman (India), or Sturtevant (USA). A jet mill uses 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.

[0040] A process that is run continuously is a process that is not run in a batch mode. When the milling process 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.

[0041] 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®).

[0042] 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).

[0043] Examples of amino acids according to the present invention are tyrosine, protein organic amino acids, especially essential amino acids, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, and non-protein organic amino acids, preferably L-amino acids. Tyrosine means L- or D-tyrosine, preferably L-tyrosine. Cysteine means L- or D-cysteine, preferably L-cysteine.

[0044] 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 B 12(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.

[0045] 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, 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.

[0046] Examples of cofactors are thiamine derivatives, biotin, vitamin C, NAD / NADP, cobalamin, flavin mononucleotide and derivatives, glutathione, nucleotides, phosphates and derivatives. According to the present invention, nucleic acid components are nucleobases 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.

[0047] 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 separate feeds for production reasons.

[0048] 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

[0049] 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).

[0050] According to the present invention, freezing means cooling to a temperature below 0°C. The gist of the present invention is that by simply mixing a powder and a solvent, a powdered cell culture medium such as a dry powder medium produced by dry compression, which can be easily dissolved in a suitable solvent, can produce a liquid cell culture medium suitable for culturing cells such as a complete medium, a medium supplement, a medium subgroup or a feed, having a desired uniform concentration of medium components by dissolving the powder.

[0051] The simple dissolution of powdered cell culture media is often complicated by substances with poor solubility in aqueous solvents, especially amino acids. For example, L-tyrosine has a solubility of 0.4 g / l in water at a temperature of 25°C. This means that approximately 0.4 g of L-tyrosine is soluble in 1 liter of water. However, the required concentration of tyrosine in cell culture media is often higher. Also, the solubility of leucine of approximately 22.1 g / kg at 25°C and that of isoleucine of 32.4 g / kg at 25°C are often considered insufficient.

[0052] On the one hand, N-lactyl derivatives of amino acids typically have high solubility in aqueous solutions, and on the other hand, they can be used as substitutions for the respective amino acids and are equally suitable as cell culture medium components as the corresponding native amino acids. Preferably, to achieve optimal performance, the cell culture medium according to the present invention contains native amino acids and N-lactyl amino acids. If the medium is a feed medium or another medium additive added to a basal medium containing native amino acids, the feed medium or medium additive may contain only N-lactyl amino acids and not the corresponding native amino acids.

[0053] Typically, in the basal medium and the perfusion medium, the molar ratio of the N-lactyl amino acid to the corresponding native amino acid is from 5:1 to 1:5, preferably from 5:1 to 1:1. The overall concentration of each lactyl amino acid in the ready-to-use liquid basal / perfusion medium and in the feed medium or medium additive is very flexible. The upper limit is defined only by the solubility of the lactyl amino acid in each medium. As a result, it is typically possible to produce a liquid medium with a lactyl amino acid concentration up to 500 mmol / l or more.

[0054] The solubility of the N-lactyl amino acid is greater than that of the respective native amino acid (see Tables 1 and 2 of Example 2). To further increase the solubility of the N-lactyl amino acid, the salt can be formed by reacting the derivative with a suitable base. Sodium salts are preferred. The cell culture medium according to the present invention may contain one or more N-lactyl amino acids, meaning for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0055] The powdered cell culture medium of the present invention is preferably produced by mixing all the components and milling them. Mixing the components is well known to those skilled in the art for producing a dry powdered cell culture medium by milling. Preferably, all the components are thoroughly mixed so that all parts of the mixture have substantially the same composition. With respect to homogeneous cell growth, the higher the homogeneity of the composition, the better the quality of the resulting medium.

[0056] Milling 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. A person skilled in the art knows how to operate such mills.

[0057] Large-scale equipment mills with a disk diameter of about 40 cm are typically run at 1 to 6500 revolutions per minute, preferably 1 to 3000 revolutions per minute, for example in the case of a pin mill. Milling can be carried out under standard milling conditions, resulting in a powder with a particle size between 10 and 300 μm, most preferably between 25 and 120 μm.

[0058] Preferably, all components of the mixture to be milled are dry. This means that if they contain water, they contain only water crystals that are not more than 10% by weight of unbound or non-agglomerated water molecules, preferably not more than 5% by weight, and most preferably not more than 2% by weight. In a preferred embodiment, milling is carried out in an inert atmosphere. A preferred inert protective gas is nitrogen.

[0059] In a further preferred embodiment, all components of the mixture are frozen prior to milling. 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 with liquid nitrogen. This means that, for example, prior to introduction into the mill, the components are treated with liquid nitrogen by pouring liquid nitrogen into the container in which the components are stored. 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.

[0060] Typically, the components are treated with liquid nitrogen for 2 to 20 seconds. Preferably, the cooling of the components is done in such a way that all the components entering the mill are at a temperature below 0 °C, most preferably below -20 °C.

[0061] In a preferred embodiment, all the components are placed in a container into which the mixture is transferred 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 cooled mixture is transferred from the feeder to the mill such that the mixture to be milled still has a temperature preferably below 0 °C, more preferably below -20 °C.

[0062] 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.

[0063] Typically, the temperature at which milling is carried out is maintained between -50 and +30 °C. In a preferred embodiment, the temperature is maintained 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 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.

[0064] After milling, the resulting dry powder medium may be further compressed, for example by dry compression with a roll press, to increase the particle size. For the use of the powdered 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, resulting in a liquid medium that can be used immediately.

[0065] The solvent may also contain physiological saline, soluble acid or base ions that provide a suitable pH range (typically in the range of pH 1.0 to pH 10.0), stabilizers, surfactants, preservatives, and alcohol or other polar organic solvents.

[0066] It is also possible to further add substances such as buffer substances for pH adjustment, fetal bovine serum, sugars, etc. to the mixture of the cell culture medium and the solvent. The resulting liquid cell culture medium is then brought into contact with the cells to be grown or maintained.

[0067] A medium composition containing a high concentration of one or more native amino acids shows turbidity when mixed with a solvent due to the undissolved amino acids, while the cell culture medium according to the present invention in which the amino acids are completely or partially replaced with the corresponding N-lactyl amino acids gives a clear solution, as shown by the turbidity measurements in Example 3 and Figures 2 and 3.

[0068] The present invention further relates to a) providing a bioreactor b) mixing the cells to be cultured with the cell culture medium according to the present invention c) incubating the mixture of step b) and is directed to a process for culturing cells. In one embodiment, the bioreactor is a perfusion bioreactor.

[0069] A bioreactor is any vessel or tank in which cells can be cultured. Incubation is typically done 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.

[0070] A perfusion bioreactor is a bioreactor in which perfusion cell culture can be carried out. It typically includes a bioreactor vessel that is closed during cell culture, a stirrer in the vessel, a line for introducing fresh medium, a harvest line for removing a harvest stream containing cells, liquid medium, and the target product from the bioreactor, and a cell retention device that can recover the liquid portion of the harvest while retaining the cells in the harvest line. A review on perfusion cell culture providing details on a preferred setup can be found in “Perfusion mammalian cell culture for recombinant protein manufacturing - A critical review” Jean-Marc Bielser et al., Biotechnology Advances 36 (2018) 1328-1340.

[0071] The present invention has been found to be extremely suitable for the preparation of feed media. Due to limitations in the availability of certain amino acids at the concentrations required in the feed medium, it has not been possible to prepare the feed medium at the desired high concentrations, or it has been necessary to prepare them under drastic pH conditions such as a very basic pH. This has an adverse effect on the nutrient supply to the cells and may to some extent promote cell death due to exposure to extreme basic pH values.

[0072] As a result, there is a need for a feed medium that contains all the required components at high concentrations in a single feed. In addition, the pH of the feed should not have an adverse effect on cell culture.

[0073] N-lactyl amino acids have improved solubility and can be used in highly concentrated feed media instead of the corresponding native amino acids, and have been found to have no adverse effect on cell growth and / or productivity at pH below 8.5, and sometimes even have a beneficial effect.

[0074] The present invention is therefore also directed to any feed medium in the form of a powdered medium or in the form of a liquid medium after dissolution. The resulting liquid medium typically contains one or more N-lactyl amino acids at a concentration of more than 10 mmol / l or more than 50 mmol / l and preferably has a pH of 8.5 or less. In a preferred embodiment, the pH is from 6.5 to 7.8.

[0075] The present invention also relates to - filling a bioreactor with cells and an aqueous cell culture medium - incubating the cells in the bioreactor - adding the cell culture medium to the bioreactor continuously over the entire time during the cell incubation time, or one or several times wherein the added cell culture medium preferably has a pH below 8.5 and contains at least one N-lactyl amino acid, and is directed to a process for culturing cells in a bioreactor. Typically, the medium contains solid components dissolved in a solvent of 50 to 150 g / l.

[0076] In one embodiment, the added medium is a feed medium and the process is a fed-batch process. In another medium, the added medium is a perfusion medium and the process is a perfusion process.

[0077] By using N-lactyl amino acids of 1 or more, it has been found that a feed medium containing all necessary feed components can be obtained at a high concentration (total concentration 100 to 250 g / l). In contrast to known processes where 2 or more different feed media are required for supply to a bioreactor, the present invention provides a medium and method that enable the use of one feed medium containing all components at a high concentration. In addition, the pH of the feed medium according to the present invention is typically less than 8.5.

[0078] In a preferred embodiment, in the process of the present invention, the feed medium added to the bioreactor continuously during incubation or one or several times within the said time always has the same composition. The present invention is further illustrated by the following figures and examples, but is not limited thereto. All applications, patents, and publications cited above and below, and the entire disclosure of the corresponding European patent application EP19209150.2 filed on November 14, 2019, are incorporated herein by reference.

Example

[0079] Example The following examples represent practical applications of the present invention. Example 1 Synthesis of L-lactyl-L-phenylalanine L-lactyl-L-phenylalanine was synthesized starting from free phenylalanine (Sforza, S., et al., Accumulation of non-proteolytic aminoacyl derivatives in Parmigiano-Reggiano cheese during ripening. International Dairy Journal, 2009. 19(10): p. 582-587). L-phenylalanine methyl ester hydrochloride was first synthesized: 2.00 g of L-phenylalanine (12.05 mmol) was dissolved in 100 mL of methanol and maintained under continuous stirring in an ice bath; SOCl 2was slowly added to a final concentration of 1 M. The reaction was monitored by TLC (eluent: n-butanol: acetic acid: H 2 O, 4:1:1 by vol; UV and ninhydrin detection, Rf 0.6) overnight. The reaction mixture was dried under vacuum, methanol was added, and evaporated again under reduced pressure (4 times) to completely remove HCl; the crude yield was 98%. (S)-2-Acetoxypropionic acid (0.41 g, 3.12 mmol) was then dissolved in 4 mL of CH 2 Cl 2 together with 1.12 g (2.96 mmol) of HBTU, and the mixture was maintained under continuous stirring at room temperature for 30 minutes to activate the carboxyl functional group. L-Phenylalanine methyl ester hydrochloride (0.68 g, 3.12 mmol) synthesized so far was dissolved in 4 mL of CH 2 Cl 2 together with DIPEA (1.54 mL, 9.36 mmol), and then added to the activated acetoxypropionic acid. The reaction was left at room temperature for 4 hours under magnetic stirring. The reaction was monitored by TLC (eluent: ethyl acetate; UV absorbance detector, Rf 0.8). The organic solution was then washed with saturated solutions of KHSO 4 (3 times) and NaHCO 3 (3 times), dried over MgSO 4 , filtered, and the product was dried under vacuum; the crude yield was 46%. The methyl and acetyl protecting groups were then removed by reacting the product in a mixture of 0.34 g (1.296 mmol) of Ba(OH) 2 ·5H 2 O in 20 mL of tetrahydrofuran (THF) / H 2 O (1:1, v / v) at 0 °C for 20 minutes. Then THF was removed under vacuum, and the aqueous solution was acidified to pH 3.0 with HCl. The resulting solution was analyzed by LC / ESI-MS.

[0080] Example 2: Lac-Ile and Lac-Leu have increased solubility compared to their respective amino acids in water The maximum solubility of Ile and Leu was compared with that of their respective lactoyl derivatives or their salts in water at 25 °C by preparing saturated solutions. After sedimentation, the solutions were dried using infrared rays (120 °C, 120 minutes), and the residual mass was determined in g / kg. As shown in Table 1, the solubilities of their lactoyl-AA and salts were significantly higher when compared with the solubility of the respective amino acids in water. In the case of Leu, the increase in solubility was approximately 30-fold with Lac-Leu (N-lactoyl leucine), while with Lac-Ile (N-lactoyl isoleucine), the increase was 20-fold when compared with Ile. To rule out that the increase in solubility is due to the sodium salt form of lactoyl-AA, separate experiments were conducted to compare the solubilities of Leu, sodium salt of Leu, and sodium salt of lac-Leu. The maximum solubilities obtained in water were 22.1, 86.0, and 689.2 g / kg, respectively. As expected, the formation of the sodium salt increases the solubility of Leu, but the increase in solubility obtained with the lactoyl derivative is significantly important, thus indicating that it cannot be attributed only to the salt form. The same behavior is considered valid for other lac-AA. Overall, these results indicate that lac-AA and their salts are suitable candidates for increasing the solubility of cell culture media and feed formulations by replacing their respective amino acids.

[0081] Table 1: Solubility in water at 25 °C of amino acids and their respective lac-AA or their salts. The solubility experiments were carried out using saturated solutions after infrared drying and determination of the residual mass.

Table 1-1

Table 1-2

[0082] Example 3: Maximum solubility of Lac-AA compared with their respective amino acids in Cellvento® 4 Feed lacking Ile and Leu Increased amounts of Lac-Leu and Lac-Ile and their salts were added to a cell culture feed formulation (Cellvento® 4Feed) lacking Ile and Leu. 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 113 g / L and the pH was 7.0 + / - 0.2. In small-scale experiments, after each addition of either amino acid or Lac-AA, the feed was stirred for 10 minutes and the turbidity was measured. The experiments were conducted at room temperature (25 °C).

[0083] The maximum solubility of Ile in Cellvento® 4Feed lacking Ile / Leu was found to be approximately 105 mM, while Lac-Ile was found to be soluble at turbidity values of less than 5 NTU even at the maximum test concentration of 951 mM (Figure 2). This indicates that in Cellvento® 4Feed lacking Ile / Leu, Lac-Ile is at least 9-fold more soluble than Ile.

[0084] Figure 2 shows the determination of the maximum solubility of Ile or Lac-Ile in a Cellvento® 4Feed formulation lacking Ile and Leu (113 g / L, pH 7.0 + / - 0.2). A solution with a turbidity of less than 5 NTU is considered soluble.

[0085] The maximum solubility of Leu in Cellvento® 4Feed lacking Ile and Leu was found to be approximately 90 mM, while for Lac-Leu, the maximum solubility concentration (turbidity value less than 5 NTU) was found to be approximately 600 mM (Figure 3). This indicates that in Cellvento® 4Feed lacking Ile / Leu, Lac-Leu is more than 6.6-fold more soluble than Leu.

[0086] Figure 3 shows the determination of the maximum solubility of Leu or Lac-Leu in the Cellvento® 4Feed formulation lacking Ile and Leu (113 g / L, pH 7.0 + / - 0.2). A solution with a turbidity of less than 5 NTU is considered soluble.

[0087] Example 4: The use of Lac-AA enables the concentration of cell culture medium formulations at neutral pH. The maximum solubility of Cellvento® 4Feed (standard concentration 130 g / L) was determined by dissolving increasing amounts of the feed dry powder medium in water until precipitation was visually detected. At 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. The osmotic pressure and turbidity were measured and images were acquired (Figure 4). The data indicate that a 1.2-fold concentrate of this formulation (160 g / L) is already insoluble as particles were detected in the suspension and the turbidity greatly exceeded the 5 NTU limit. Figure 4 shows the solubility limit of Cellvento® 4Feed at pH 7.0. Turbidity was measured using a turbidimeter.

[0088] 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 (Cellvento® 4Feed-Ile / Leu). The maximum concentration of this feed supplemented with Lac-Leu and Lac-Ile was determined by dissolving increasing amounts of the feed dry powder medium + lactoyl derivative 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.

[0089] The results show that the maximum solubility of Cellvento® 4Feed lacking Ile / Leu supplemented with Lac-Leu and Lac-Ile was obtained at 189 g / L to 212 g / L (Figure 5). Considering that Cellvento® 4Feed (containing Ile and Leu) has a concentration of 130 g / L, this represents an approximate 50% increase in concentration when Ile and Leu are replaced by Lac-Ile and Lac-Leu.

[0090] Figure 5 shows the turbidity of solutions containing increasing amounts of 4Feed lacking Ile / Leu and supplemented with Lac-Leu and Lac-Ile derivatives (equimolar concentrations compared to free AA).

[0091] Example 5: When Cellvento 4Feed-Ile / Leu was stored for 3 months at either 4°C or RT protected from light, Lac-Leu and Lac-Ile were stable To monitor the stability of Lac-AA in complex feed mixtures, a targeted quantitative LC-MS method was developed. Serial dilutions of Lac-Leu and Lac-Ile were performed from 100 mM to 100 μM in Cellvento® 4Feed-Ile / Leu at pH 7.0 to determine the linearity of the method. Prior to LC-MS analysis, a 200-fold dilution with water was performed. The method was developed on a UHPLC (Vanquish, Thermo Fisher) combined with an ESI-Q-ToF mass spectrometer (Impact II, Bruker Daltonics). Briefly, 1 μL of sample was loaded at a flow rate of 300 μL / min onto a XSelect HSS T3 column (2.1 x 150 mm, 3.5 μm, Waters) temperature-controlled at 40°C in 99.9% buffer A (20 mM ammonium formate / 0.1% FA) and eluted with a multi-step gradient of buffer B (100% methanol) presented in Table 2.

[0092]

Table 2

[0093] LC-MS analysis was performed using an Impact II mass spectrometer (Bruker Daltonics) equipped with an ESI source. MS capture was performed in negative mode with an end plate, and the offset voltage and capillary voltage were set to 500 V and 3500 V, respectively. The nebulizer and drying gas (250 °C) were set to 1.4 bar and 9.0 L / min, respectively. The MS spectrum was acquired over the m / z range of 20 - 1000 at a scan rate of 5 Hz. Calibration was performed using the sodium formate solution injected at the start of the analysis. The obtained standard curves for Lac-Leu and Lac Ile are shown in Figure 6. Extremely good linearity was obtained with injections on the column in the range of 10 pmol - 400 pmol (500 μM - 20 mM in the feed), while it was not obtained in the range of 2 pmol - 2 nmol (100 μM - 100 mM in the feed, not shown) on the column. Therefore, Lac-AA quantification should be performed by injecting 10 - 400 pmol on the column.

[0094] Figure 6 shows the linearity obtained by the LC-MS method after 200× dilution with water for Lac-Leu and Lac-Ile in Cellvento® 4Feed-Ile / Leu. Extremely good linearity was obtained in the range of 10 - 400 pmol on the column.

[0095] Using the method developed so far, the stability of Lac-Ile and Lac-Leu in Cellvento® 4Feed-Ile / Leu was monitored in samples stored for 3 months in the light, at 4 °C, and at RT. As shown in the figure, the stability of both lactoyl AAs is extremely good at 4 °C and RT, and Lac-Ile and Lac-Leu were shown to be stable when the feed formulation was protected from light and stored at either 4 °C or RT for up to 3 months. Figure 7 shows the stability of Lac-Ile and Lac-Leu in Cellvento® 4Feed-Ile / Leu as determined by LC-MS.

[0096] Example 6: Lac-Ile and Lac-Leu can replace their respective amino acids in the feed. Cell culture results with a CHOK1GS clone producing IgG1. For the cell culture experiments, 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 2 x 10 5 cells / mL. Incubation was carried out at 37 °C, 5% CO2, 80% humidity, and agitation at 320 rpm. Lac-AA was added to the feed (Cellvento® 4Feed lacking Ile and leu) in place 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 the respective amino acids without addition of Lac-AA. Feeding was done on days 3, 5, 7, 10, 12, and 14 at the following v / v ratios (3, 3, 6, 3, 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.

[0097] Viable cell density (VCD) and viability were evaluated using a Vi-CELL XR (Beckman Coulter, Fullerton, CA). Metabolite concentrations were monitored using a Cedex Bio HT (Roche Diagnostics, Mannheim, Germany) based on spectrophotometry and turbidimetry. Amino acid quantification was performed by UPLC after derivatization using an AccQ·TagUltra® reagent kit. Derivatization, chromatography, and data analysis were performed according to the recommendations of the supplier (Waters, Milford, MA).

[0098] Considering the viable cell density (Figure 8), the negative control with the feed lacking Leu and Ile showed a rapid decrease in VCD after 7 days, indicating that cells require both amino acids for efficient growth. Replacement of Ile with Lac-Ile had no effect on the VCD peak or the overall VCD profile compared to the positive control, while replacement of Leu with Lac-Leu had a positive effect during the plateau period, with the cells remaining at approximately 15 million cells / ml for several days from day 10 to day 14.

[0099] Figure 8 shows the VCD over a 17-day fed-batch process with each of Leu and Ile in the feed replaced with either Lac-Leu or Lac-Ile. The depleted Cellvento® 4Feed is the negative control and contains no Leu or Ile. The IgG concentration obtained on day 14 was approximately 3 g / L under all conditions (Figure 9), and was slightly higher at a non-significant titer under the conditions where Leu and Ile were replaced with Lac-Leu and Lac-Ile, respectively.

[0100] Figure 9 shows the IgG produced over a 17-day fed-batch process with each of Leu and Ile in the feed replaced with either Lac-Leu or Lac-Ile. The formation of free AA from Lac-AA releases lactate, and this metabolite was monitored in the supernatant during the FB process (Figure 10). High lactate concentrations were observed after 5 days under the conditions where Leu and Ile were replaced with Lac-Leu and Lac-Ile, respectively, which could be due to the cleavage of the derivatives. When calculating the area under the curve of the lactate concentration, the overall increase in lactate was quantified. Replacement of Leu with Lac-Leu resulted in a 20% increase in free lactate, while replacement of Ile with Lac-Ile resulted in a 37% increase, which was very similar to the increase in lactate (36.5%) observed in the negative control.

[0101] Figure 10 shows lactate production during a 17-day fed-batch process in which each of Leu and Ile in the feed was replaced with either Lac-Leu or Lac-Ile. The concentration of amino acids in the spent medium was determined. Under the condition where Leu was replaced with Lac-Leu, the concentration of Leu in the spent medium (Figure 11) decreased very rapidly until day 7 and then increased again, suggesting that the cleavage of Lac-AA takes time and likely depends on the release (a sustained-release technique similar to the phosphotyrosine release technique developed so far) or activation of specific enzymes. Under the condition where Ile was replaced with Lac-Ile, the overall leucine concentration decreased moderately over the fed-batch, indicating that other branched-chain amino acids can be used in place of Ile before efficient cleavage of the lactoyl derivative.

[0102] Considering Ile in the spent medium (Figure 12), behavior similar to that of Lac-Leu was detected. Under the condition where Ile was replaced with Lac-Ile, the concentration of Ile in the spent medium decreased very rapidly until day 7 and then increased again, suggesting that the cleavage of Lac-AA takes time and likely depends on the release or activation of specific enzymes. Under the condition where Leu was replaced with Lac-Leu, the Ile concentration decreased after 7 days, also indicating that other branched-chain amino acids can be used in place of Leu.

[0103] Figure 11: Leu quantification in the spent medium during a 17-day fed-batch process in which each of Leu and Ile in the feed was replaced with either Lac-Leu or Lac-Ile. Figure 12: Ile quantification in the spent medium during a 17-day fed-batch process in which each of Leu and Ile in the feed was replaced with either Lac-Leu or Lac-Ile.

[0104] 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 the feed lacking either Leu or Ile and supplemented with either Lac-Leu or Lac-Ile.

[0105] The antibody was purified from the cell culture supernatant 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 pattern was analyzed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF). Briefly, the purified antibody was denatured and immobilized, digested with N-Glycanase®, and the glycans were released from the antibody by labeling with APTS at 50 °C for 60 minutes. After a washing step to remove the remaining APTS, the relative amounts of the glycans were determined using a Pharmaceutical Analysis System CESI8000 Plus (Sciex, Washington, USA) equipped with an LIF detector (Ex: 488 nm, Em: 520 nm).

[0106] Separation was carried out in a polyvinyl alcohol-coated capillary (total length: 50.2 cm, inner diameter: 50 μm), which was filled with a carbohydrate separation buffer from a carbohydrate labeling kit (Beckman Coulter, Brea, USA). The capillary surface was first rinsed with the separation buffer at 30 psi for 3 minutes. The buffer vials at the inlet and outlet were exchanged every 20 cycles. The sample was introduced by pressure injection at 0.5 psi for 12 seconds, followed by an immersion step for 0.2 minutes 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.

[0107] 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.

[0108] 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.

[0109] The results obtained for glycosylation (Figure 13), high molecular weight and low molecular weight species (Figure 14), and charge variants (Figure 15) showed no difference between the control conditions and those in which Ile and Leu were replaced with Lac-Ile and Lac-Leu, indicating that the amino acid exchange did not affect three important quality attributes of the IgG1 produced in this study.

[0110] Figure 13 shows the glycosylation of IgG1 produced in a process using a control process or a feed lacking Ile / Leu and supplemented with either Lac-Leu or Lac-Ile. The glycoform distribution was determined using APTS labeling and CGE-LIF detection.

[0111] Figure 14 shows the aggregation and fragmentation of IgG1 produced in a process using a control process or a feed lacking Ile / Leu and supplemented with either Lac-Leu or Lac-Ile. High molecular weight (HMW) and low molecular weight species (LMW) were determined using size exclusion chromatography.

[0112] Figure 15 shows the charge variants of IgG1 produced in a process using a control process or a feed lacking Ile / Leu and supplemented with either Lac-Leu or Lac-Ile. The charge variant distribution was determined using cIEF on a capillary electrophoresis CESI 8000.

[0113] Example 7: Confirmation of Lac-Leu and Lac-Ile performance by the CHODG44 clone producing IgG1. Fed-batch experiments were carried out using the CHODG44 clone to demonstrate the applicability of the present invention to various bioprocesses. The results (Figs. 16, 17, 18) show similar VCD and IgG titers for the Lac-Leu and Lac-Ile conditions compared to the control positive control. In contrast, the depleted Cellvento® 4Feed condition led to an early decrease in VCD and a significantly reduced titer after 7 days. Data on the spent medium did not detect an increase in lactate from the cleavage of the Lac derivative for this cell line. The explanation for this difference in behavior between CHOK1GS cells and this DG44 cell line is currently unknown. The concentrations of Leu and Ile in the spent medium (Figs. 19 and ) decreased during the first day of FB culture and finally increased again after D7. This behavior is similar to that seen in CHOK1GS cells and indicates a slow-release process of free AA from the derivative.

[0114] Figs. 16, 17, and 18 show the performance of the Lac-Leu and Lac-Ile processes compared to the control for the CHODG44 cell line expressing IgG1. Viable cell density (Fig. 16), IgG titer (Fig. 17), lactate concentration (Fig. 18) in the spent medium. Figs. 19 and 20 show the relative concentrations of isoleucine (Fig. 19) and leucine (Fig. 20) in the spent medium in the Lac-Leu and Lac-Ile processes (CHODG44 cells) compared to the normal process using unmodified Ile and Leu.

Claims

A cell culture medium comprising at least one N-lactyl-amino acid and / or a salt thereof, characterized in that it is a dry powder medium. Claim 2 The cell culture medium according to claim 1, characterized in that the N-lactyl-amino acid is selected from N-lactyl-leucine, N-lactyl-isoleucine, N-lactyl-valine, N-lactyl-phenylalanine, N-lactyl-tyrosine and / or N-lactyl-methionine. The cell culture medium according to claim 1, characterized in that the N-lactyl-amino acid is selected from N-lactyl-leucine and / or N-lactyl-isoleucine. Claim 4 The cell culture medium 【Chemical 1】 wherein, R １ ＋ is H ＋ or Na ＋ or K ＋ or Mg ２＋ or Ca ２＋ or Li ＋ and R ２ is as follows: [Chemical Formula 2] is selected from one or more of formula I: The cell culture medium according to any one of claims 1 to 3, characterized in that it contains a component of. Claim 5 The cell culture medium according to any one of claims 1 to 4, characterized in that the cell culture medium contains a sodium salt of one or more N-lactyl amino acids. Claim 6 The cell culture medium according to any one of claims 1 to 5, characterized in that the cell culture medium contains one or more N-lactyl amino acids and the corresponding amino acids and / or salts thereof. Claim 7 The cell culture medium according to any one of claims 1 to 6, characterized in that the medium contains one or more N-lactyl amino acids and / or salts thereof, but does not contain the corresponding amino acids and / or salts thereof. Claim 8 a) Mixing one or more N-lactyl-amino acids according to formula I with other components of the cell culture medium b) Subjecting the mixture of step a) to milling A method for producing the cell culture medium according to any one of claims 1 to 7. Claim 9 a) Providing a bioreactor b) Mixing the cells to be cultured with the cell culture medium according to any one of claims 1 to 7 c) Incubating the mixture of step b) A process for culturing cells. Claim 10 The process according to claim 9, characterized in that the bioreactor is a perfusion bioreactor. Claim 11 - Filling the bioreactor with cells and an aqueous cell culture medium - Incubating the cells in the bioreactor - adding the cell culture medium to the bioreactor continuously over the entire duration of the cell incubation time, or once or several times wherein the medium is the cell culture medium according to any one of claims 1 to 7 for culturing cells in a bioreactor. **Claim 12** **Claim 13** The process according to claim 11, wherein the process is a fed-batch process and the added medium is a feed medium having a pH of less than 8.5 and containing at least one N-lactyl amino acid and / or a salt thereof at a concentration of more than 10 mmol / l. The process according to claim 11 or 12, wherein the N-lactyl amino acid is N-lactyl leucine and / or N-lactyl isoleucine and / or a salt thereof.

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