Cell culture medium

Abstract

The present invention relates to a cell culture medium comprising N-norvalyl-L-tyrosine. The poor solubility of tyrosine in cell culture media is overcome by partially or totally replacing it with N-norvalyl-L-tyrosine.

Description

[Technical Field]

[0001] The present invention relates to a cell culture medium comprising N-norvalyl-L-tyrosine. The poor solubility of tyrosine in cell culture media is overcome by partially or totally replacing it with N-norvalyl-L-tyrosine. [Background technology]

[0002] Cell culture media supports and maintains the growth of cells in an artificial environment. Depending on the type of organism whose growth is being supported, cell culture media contain a complex mixture of components, sometimes over 100 different components. The cell culture media required for the growth of mammalian, insect or plant cells are typically much more complex than media to support bacterial and yeast growth. The first cell culture media developed consisted of undefined components such as plasma, serum, embryonic extracts, or other undefined biological extracts or peptones. Major advances have been made with 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 other substances known to those skilled in the art.

[0003] Some cell culture media are proposed as sterile aqueous liquids. The disadvantages of liquid cell culture media are their short shelf life and the difficulty of transporting and storing them. As a result, many cell culture media are currently proposed as finely ground dry powder mixtures. These are manufactured for dissolution in water and / or aqueous solutions, and in the dissolved state, are often designed, together with other supplements, to replenish cells with a substantial nutrient base for the growth and / or production of biopharmaceuticals from said cells.

[0004] Many biopharmaceutical production platforms are based on fed-batch cell culture protocols. The goal is typically to develop high-titer cell culture processes that meet growing market demands and reduce manufacturing costs. In addition to fed-batch, perfusion is also a common culture method for biopharmaceutical production in N-stage bioreactors, but also for N-1 bioreactors. The goal is typically to shorten seed train times by achieving higher cell densities and higher volumetric productivity while maintaining high viability. Achieving maximum production capacity requires the use of high performance recombinant cell lines as well as improvements in cell culture media and process parameters.

[0005] In a fed-batch process, a basal medium supports initial growth and production, and a feed medium prevents nutrient starvation and sustains the production phase. Media are selected to accommodate distinct metabolic requirements during the various production phases. The set of process parameters, including feeding strategies and control parameters, defines the chemical and physical environment favorable for cell growth and protein production. In the perfusion process, the basal medium supports initial growth and production up to a certain point (up to the exponential growth phase, without nutrient limitation), where medium is simultaneously added and removed from the bioreactor at a specific medium exchange rate, which is the bioreactor workload per day (VVD, d -1 ) perfusion rate (P, d -1 It can be described either by the daily perfusion rate (CPR) or the cell-specific perfusion rate (CSPR, pL / cell / d). The CSPR displays the nutrient supply per cell and per day in a continuous process. Therefore, the CSPR is cell line and medium specific.

[0006] Optimization of the feed medium is a major aspect in the optimization of fed-batch processes. In most cases, the feed medium is highly concentrated to avoid dilution of the product (antibody or recombinant protein) in the bioreactor. The controlled addition of nutrients directly affects the growth rate and lifespan of the culture. For continuous processes, optimization of the perfusion medium and minimization of CSPR are key aspects. It is well known from the literature that concentrated medium can be used to reduce CSPR.

[0007] Amino acids (AA), such as tyrosine, are essential components of cell culture media because they are important for supporting cell growth. Additionally, AA is a key building block of recombinant proteins produced using mammalian cell culture techniques. The solubility of tyrosine is a limiting factor that prevents 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 needed for biomanufacturing processes that use in-line dilution to reduce the volume of CCM that must be stored in tanks (i.e., reduce the manufacturing footprint) or, in general, to a. reduce the volume of feed added through fed-batch (FB) processes or b. reduce CSPR by concentrating the medium in continuous processes, thereby potentially increasing the volumetric titer. Consequently, it is desirable to find ways to improve the solubility of tyrosine.

[0008] Another important characteristic of amino acid derivatives, especially when used in culture media, is that they must be readily available to cells to support metabolic requirements. Many amino acid derivatives are not readily bioavailable and require the release of enzymes to cleave them, thereby releasing the bioavailable, natural amino acid. As a result, many highly soluble amino acid derivatives may not be suitable for cell culture media.

[0009] US2019 / 0390161 discloses a method for culturing cells using a cell culture medium containing at least one oligopeptide of 2 to 10 amino acids, wherein these amino acids are natural and selected from the following amino acids: Cys, Cyss, Leu, Tyr, Val, Ile. WO2012 / 019160 discloses a method for culturing CHO cells using a serum-free defined production medium and feed supplemented with specific dipeptides, including the Tyr-containing dipeptides Tyr-His, Tyr-Lys, Tyr-Ala, and Tyr-Val.

[0010] It has been unexpectedly discovered that tyrosine can be substituted with N-norvalyl-L-tyrosine or its salts in cell culture media. In addition to its use as a tyrosine source, N-norvalyl derivatives exhibit higher solubility than tyrosine and can therefore be used in highly concentrated formulations. In addition, compared to other tyrosine derivatives, N-norvalyl derivatives are readily available to cells, ensuring a continuous supply of tyrosine necessary for growth and recombinant protein production. Thus, the present invention is directed to a cell culture medium comprising N-norvalyl-L-tyrosine. Hereinafter, when this component is referred to, it refers to the free dipeptide, as well as the hydrochloride salt, Na + , K. + , Mg 2+ , Ca 2+ , Li + Those skilled in the art will recognize that either the free dipeptide or any of its salts can be used.

[0011] In a preferred embodiment, the cell culture medium comprises the hydrochloride salt of N-norvalyl-L-tyrosine. In a preferred embodiment, the cell culture medium is a dry powder medium. In one embodiment, particularly when the cell culture medium is a basal medium or a perfusion cell culture medium, it comprises N-norvalyl-L-tyrosine, and optionally native tyrosine, where native means the unmodified amino acid and / or its salt. In another preferred embodiment, the cell culture medium is a feed medium. The feed medium may contain N-norvalyl-L-tyrosine and the corresponding native tyrosine, but it may also contain only N-norvalyl-L-tyrosine and no corresponding native tyrosine.

[0012] In a preferred embodiment, the cell culture medium contains N-norvalyl-L-tyrosine but does not contain tyrosine. "Does not contain tyrosine" means that it does not contain any L-tyrosine and / or a salt thereof, or that it contains L-tyrosine and / or a salt thereof at a molar ratio of less than 1 / 10 (L-tyrosine and / or a salt thereof / N-norvalyl-L-tyrosine). In another preferred embodiment, the cell culture medium is a liquid medium having a pH of 8.5 or less and containing N-norvalyl-L-tyrosine at a concentration of at least 1 mmol / L. In the case of a feed medium, the concentration is typically greater than 10 mmol / L. The upper limit is defined only by the solubility of N-norvalyl-L-tyrosine. Solubility may depend on the solvent, pH, and salt concentration. As a result, it is typically possible to produce a liquid medium with an N-norvalyl-L-tyrosine concentration of up to 100 mmol / L.

[0013] In a preferred embodiment, the pH of the liquid medium is 6.0 to 8.5, and most preferably 6.5 to 7.8. In one embodiment, the medium is in an x-fold concentrated form relative to the concentration of said medium in use, where x is between 1.5 and 100, for example, 2-fold, 3-fold, 4-fold, 5-fold or 10-fold concentrated form. In one embodiment, the cell culture medium comprises 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.

[0014] The present invention further comprises: a) mixing N-norvalyl-L-tyrosine with other components of a cell culture medium, wherein the components are in a dry state; b) subjecting the mixture of step a) to grinding; The present invention is directed to a method for producing a cell culture medium according to the present 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 grinding.

[0015] The present invention further comprises: a) providing a bioreactor; b) optionally, producing a liquid cell culture medium according to the present invention by dissolving a dry powder medium according to the present invention in an aqueous liquid; this step is required when the cell culture medium containing N-norvalyl-L-tyrosine is a dry powder medium, but is not required when the medium is already in the form of a liquid medium; c) mixing the cells to be cultured with the liquid cell culture medium according to the invention in said bioreactor; d) incubating the mixture of step b); is directed to a process for culturing cells.

[0016] In one embodiment, the bioreactor is a perfusion bioreactor. In a preferred embodiment, the process for culturing cells is a process for perfusion cell culture comprising culturing cells in a perfusion bioreactor equipped with a medium inlet and a harvest outlet, wherein i. During the cell culture process, continuously or once or several times, fresh cell culture medium according to the present invention is inserted into the bioreactor via the medium inlet; ii. Harvest is removed from the bioreactor via the harvest outlet, either continuously or once or several times during the cell culture process. Preferably, during the perfusion phase, fresh cell culture medium containing N-norvalyl-L-tyrosine is continuously inserted into the bioreactor via the medium inlet and harvest is continuously removed from the bioreactor.

[0017] The present invention also provides - loading the cells and aqueous cell culture medium into a bioreactor; - incubating the cells in a bioreactor; - adding cell culture medium, in this case a feed medium, to the bioreactor continuously throughout the entire cell incubation period or once or several times; wherein the feed medium is a liquid cell culture medium according to the present invention comprising at least N-norvalyl-L-tyrosine; This paper is 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 N-norvalyl-L-tyrosine at a concentration of at least greater than 10 mmol / L. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows the maximum solubility of tyrosine and norvalyl-L-tyrosine derivatives in MQ water at pH 7.3 at 25°C. See Example 1 for further details. [Figure 2] Figure 2 shows the solubility of Tyr 2Na salt 2H2O and its respective norvalyl-L-tyrosine derivative or its salt in 6x concentrated CCM (pH 7.3±0.1) at 25°C. Solubility was assessed by dissolving increasing amounts of tyrosine derivative in 6x concentrated medium and measuring turbidity. Solutions with a turbidity of less than 5 NTU are considered soluble. For further details, see Example 2. [Figure 3]Figure 3 shows the measurement of maximum solubility of Tyr 2Na salt 2H2O and NL-NVa-L-Tyr HCl in a 6-fold concentrated modified EX-CELL® Advanced HD Perfusion Medium formulation that is Tyr-depleted. For further details, see Example 3.

[0019] [Figure 4] Figure 4 shows the stability of 6x concentrated CCM containing Tyr 2Na salt 2H2O or NL-NVa-Tyr HCl analyzed by UPLC. For further details, see Example 4. [Figure 5] Figure 5 shows the cell performance of CHOK1 GS clone A in a batch process. For further details, see Example 5. [Figure 6] Figure 6 shows the cell performance of CHOK1 non-GS clones in a batch process. For further details, see Example 6. [Figure 7] Figure 7 shows the cell performance of CHOZN GS clone A in a batch process. For further details, see Example 7.

[0020] [Figure 8] Figure 8 shows the cell performance of CHOZN GS clone B in batch culture. For further details, see Example 8. [Figure 9] Figure 9 shows the cell performance of CHOZN GS clone C in batch culture. For further details, see Example 9. [Figure 10] Figure 10 shows the cell performance of CHOZN GS clone D in batch culture. For further details, see Example 10. [Figure 11] Figure 11 shows the cell performance of CHOZN GS clone E in batch culture. For further details, see Example 11.

[0021] [Figure 12]Figures 12, 13 and 14 show the characteristics of the recombinant proteins. For further details, see Example 12. [Figure 13] Same as above [Figure 14] Same as above [Figure 15] Figures 15, 16, 17 and 18 show the cell performance of CHOZN GS Clone D, CHOZN GS Clone F, CHO DG44, and CHOZN GS Clone G in dynamic perfusion culture. For further details, see Example 13. [Figure 16] Same as above [Figure 17] Same as above [Figure 18] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0022] N-norvalyl-L-tyrosine is a dipeptide in which the non-proteinogenic amino acid norvaline is covalently attached to tyrosine via a peptide bond. N-norvalyl-L-tyrosine is a product obtained by, for example, chemical or biological, e.g., enzymatic, synthesis. Typically, it is produced by chemical synthesis, e.g., according to known methods for dipeptide synthesis.

[0023] An exemplary synthesis of D- / L-norvalyl-L-tyrosine is given in Abderhalden, E., & Bahn, A. (1930). A comparative study of the effect of structure on the degradability of homologous dipeptides of L-tyrosine and related acyl halides by alkali, erepsin, and trypsin kinase was performed. a) Synthesis of DL-α-bromo-n-valine acid-L-tyrosine ester by reaction of L-tyrosine ethyl ester with DL-α-bromo-n-valine acid chloride b) Hydrolysis of DL-α-bromo-n-valeryl-L-tyrosine ester followed by evaporation c) Amination of DL-α-bromo-n-valeryl-L-tyrosine with ammonia, followed by evaporation d) Purification Fermentforschung, 11, 224.

[0024] N-norvalyl-L-tyrosine can be either NL-norvalyl-L-tyrosine or ND-norvalyl-L-tyrosine, or a mixture thereof. For most cells, the choice of either NL-norvalyl-L-tyrosine or ND-norvalyl-L-tyrosine, or a mixture thereof, does not make a difference. However, in the case of undesirable effects of one of the isomers, those skilled in the art can easily choose to prepare the cell culture medium used in accordance with the present invention with only one isomer.

[0025] As an example, NL-norvalyl-L-tyrosine, also known as (2S)-2-[[(2S)-2-aminopentanoyl]amino]-3-(4-hydroxyphenyl)propanoic acid, is shown in Formula I. [ka] The N-norvalyl-L-tyrosine according to the present invention can be, for example, Na + , K. + , Mg 2+ , Ca 2+ , Li + any salt such as, preferably Na + , most preferably the HCl salt thereof, as shown in Formula II for NL-norvalyl-L-tyrosine. [ka]

[0026] A cell culture medium according to the present invention is any mixture of components that maintain and / or support the in vitro growth of cells, such as mammalian, insect, or plant cells, as well as bacteria and yeast. It can be a complex medium or a chemically defined medium. A cell culture medium can contain all components necessary to maintain and / or support the in vitro growth of cells, or it can contain only some components so that additional components can be added separately. Examples of cell culture media according to the present invention are complete media containing all components necessary to maintain and / or support the in vitro growth of cells, and medium supplements or feeds. In preferred embodiments, the cell culture medium is a complete medium or feed medium. Complete media, also called basal media, typically have a pH of 6.5 to 7.8. Feed media preferably have a pH below 8.5, preferably 6.0 to 8.5. Basal media can be media used for batch, fed-batch, or perfusion cell culture. Typically, the cell culture medium according to the present invention is used to maintain and / or support the growth of cells in a bioreactor.

[0027] A feed or feed medium is a cell culture medium that is added at a later stage to sustain the production phase, rather than a basal medium that supports initial growth and production in a cell culture, to prevent nutrient depletion and maintain the production phase. A feed medium may have higher concentrations of some components than a basal culture medium. For example, some components, such as nutrients including amino acids or carbohydrates, may be present in a feed medium at about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or about 1000X the concentration in the basal medium.

[0028] However, it may also be present in other media than the feed medium and may be used in concentrated form. For example, a concentrated perfusion medium is a cell culture medium that supports growth and production after the initial growth and production phase once medium exchange, and therefore perfusion, is initiated. Concentrated perfusion medium can be used to reduce CSPR, thereby increasing volumetric productivity. Concentrated perfusion medium can be highly concentrated compared to basal culture medium. Concentrated perfusion medium may have a concentration >1 to even about 100 times that of basal culture medium.

[0029] Thus, the medium according to the present invention can have a concentration equivalent to the concentration during use, which is the concentration of the medium in the bioreactor in contact with the cells. The medium can also be in a concentrated form. Such concentrates can be used for storage and diluted by offline or in-line dilution before being added to the bioreactor. The concentrated medium can also be added to the bioreactor as a concentrate without further dilution. This is often the case with concentrated feed medium used in fed-batch processes or concentrated perfusion medium that is added to the perfusion bioreactor continuously or once or several times during the perfusion phase.

[0030] The culture medium of the present invention can therefore be in a concentrated form, for example, 2x, 3x, 4x, 5x, 10x, 20x, 50x, or 100x concentrated form (relative to the in-use concentration that supports cell growth and product formation in a bioreactor in contact with the cells). When such a concentrated medium is used to prepare a culture medium for use, the concentrated culture medium is diluted with an aqueous solvent, preferably sterile water. The concentrated medium according to the present invention can be a liquid medium or a dry powder medium, where the dry powder medium is provided with instructions on how much aqueous solvent should be used to dissolve the dry powder medium to produce the concentrated liquid medium. The concentrations of medium components provided herein always refer to the concentration in the respective liquid medium, where those skilled in the art will recognize that the dry powder medium will dissolve in a specific amount of aqueous solvent to provide a specific concentration of the component in the respective liquid medium.

[0031] Mammalian cell culture media is a mixture of components that maintain and / or support the growth of mammalian cells in vitro. Examples of mammalian cells are human or animal cells, preferably CHO cells, COS cells, IVERO cells, BHK cells, AK-1 cells, SP2 / 0 cells, L5.1 cells, hybridoma cells or human cells.

[0032] A chemically defined cell culture medium is a cell culture medium that does not contain any chemically undefined substances. This means that the chemical composition of all chemicals used in the medium is known. Chemically defined media do not contain any yeast, animal, or plant tissue; they do not contain feeder cells, serum, extracts, or digests, or other components that may contribute to chemically incompletely defined proteins in the medium. Chemically undefined or incompletely defined chemical components are those whose chemical composition and structure are unknown, exist in varying compositions, or can only be defined through extensive experimental effort comparable to evaluating the chemical composition and structure of proteins such as albumin or casein.

[0033] Powdered cell culture media, or dry powder media, are typically cell culture media resulting from milling, freeze-drying, or dry or wet granulation processes. This means that powdered cell culture media are granular, particulate media, not liquid media. The term "dry powder" may be used interchangeably with the term "powder." However, as used herein, "dry powder" simply refers to the overall appearance of the granular material and is not intended to imply that the material is completely free of complexed or aggregated solvents, unless otherwise indicated. Dry powder media resulting from milling or freeze-drying processes typically have a particle size of less than 0.5 mm, e.g., 0.05-0.5 mm.

[0034] Dry powder media obtained from dry or wet granulation processes, e.g., spray drying, wet granulation, or dry compaction, typically have particle sizes greater than 0.5 mm, e.g., 0.5 to 5 mm. Dry compaction is typically performed using a roll press. US Pat. No. 6,383,810 B2 discloses a method for producing agglomerated eukaryotic cell culture medium powder. The method includes wetting a dry powdered cell culture medium with a solvent and then re-drying the wetted medium to obtain a dry agglomerated cell culture medium. In one embodiment, the dry powder medium according to the present invention is produced by dry pressing.

[0035] The cells cultured using the media according to the invention may be prokaryotic cells, such as bacterial cells, or eukaryotic cells, such as plant or animal cells. The cells may be normal, immortalized, abnormal, transformed, mutated, somatic, germ, stem, progenitor, or embryonic cells, any of which may be established or transformed cell lines, or may be obtained from natural sources.

[0036] Particle size refers to the diameter of the particle. When a particle size is given, it means that at least 80%, preferably at least 90%, of the particles have the given particle size or fall within the given particle size range. Particle diameter is determined by laser light scattering. The inert atmosphere is created by filling each container or apparatus with an inert gas. Suitable inert gases are argon or preferably noble gases such as nitrogen. These inert gases are non-reactive and prevent undesired chemical reactions from occurring. In the process according to the present invention, the creation of an inert atmosphere means that the oxygen concentration is reduced to an absolute value of less than 10% (v / v), for example by introducing liquid nitrogen or nitrogen gas.

[0037] Various types of mills are known to those skilled in the art. Pin mills, also known as centrifugal impact mills, pulverize solids by providing the breaking energy through protruding pins on a rapidly rotating disk, such as those sold by Munson Machinery (USA), Premium Pulman (India), or Sturtevant (USA). Jet mills use compressed gas to accelerate particles and cause them to collide with each other in a process chamber. Jet mills are sold, for example, by Sturtevant (USA) or PMT (Austria). The Fitzmill, marketed by Fitzpatrick (USA), uses a rotor with blades to grind. A continuously run process is one that is not run batchwise. When a milling process is run continuously, it means that the media components are constantly and steadily fed to the mill over a predetermined period of time.

[0038] Cell culture media according to the invention, particularly complete media, typically include at least one or more sugar components, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffer components, one or more cofactors, and one or more nucleic acid components. The medium may also contain surface-active components such as sodium pyruvate, insulin, vegetable proteins, fatty acids and / or fatty acid derivatives and / or pluronic acid and / or chemically prepared non-ionic surfactants. One example of a suitable non-ionic surfactant is a difunctional block copolymer surfactant terminated with primary hydroxyl groups, also called poloxamer, available, for example, from BASF, Germany under the trade name pluronic®. The sugar component can be any monosaccharide or disaccharide, such as glucose, galactose, ribose, or fructose (examples of monosaccharides), or sucrose, lactose, or maltose (examples of disaccharides).

[0039] Examples of amino acids according to the invention are tyrosine, the proteinogenic amino acids, in particular the essential amino acids leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, and non-proteinogenic amino acids, preferably L-amino acids. Tyrosine means L- or D-tyrosine, preferably L-tyrosine. Cysteine ​​means L- or D-cysteine, preferably L-cysteine. Examples of vitamins are vitamin A (retinol, retinal, various retinoids and four carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxamine, pyridoxal), vitamin B7 (biotin), vitamin B9 (folic acid, folinic acid), vitamin B12 (cyanocobalamin, hydroxycobalamin, methylcobalamin), vitamin C (ascorbic acid), vitamin D (ergocalciferol, cholecalciferol), vitamin E (tocopherol, tocotrienol), and vitamin K (phylloquinone, menaquinone). Vitamin precursors are also included.

[0040] Examples of salts are compounds containing inorganic ions such as bicarbonate, calcium, chloride, magnesium, phosphate, potassium, and sodium, or trace elements such as Co, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Ni, Bi, V, and Zn. Examples are copper(II) sulfate pentahydrate (CuSO4·5H2O), sodium chloride (NaCl), calcium chloride (CaCl2·2H2O), potassium chloride (KCl), iron(II) sulfate, anhydrous monobasic sodium phosphate (NaH2PO4), anhydrous magnesium sulfate (MgSO4), anhydrous dibasic sodium phosphate (Na2HPO4), magnesium chloride hexahydrate (MgCl2·6H2O), zinc sulfate heptahydrate.

[0041] Examples of buffers are CO2 / HCO3 (carbonate), phosphate, HEPES, PIPES, ACES, BES, TES, MOPS, and TRIS. Examples of cofactors are thiamine derivatives, biotin, vitamin C, NAD / NADP, cobalamin, flavin mononucleotides and derivatives, glutathione, nucleotides, phosphates and derivatives. According to the present invention, nucleic acid building blocks are nucleic acid bases such as cytosine, guanine, adenine, thymine, or uracil, nucleosides such as cytidine, uridine, adenosine, guanosine, and thymidine, and nucleotides such as adenosine monophosphate or adenosine diphosphate or adenosine triphosphate. Feed media may have a different composition compared to complete media. They typically contain amino acids, trace elements, and vitamins. They may also contain a sugar component, although sometimes for production reasons the sugar component is added in a separate feed. A suitable feed medium may, for example, contain one or more of the following compounds:

[0042] [Table A]

[0043] According to the present invention, freezing means cooling to a temperature below 0°C. The gist of the present invention is to provide an efficient cell culture medium, on the one hand, to provide a sufficiently concentrated formulation required for the biomanufacturing process, for example using in-line dilution, to reduce the amount of CCM that must be stored in tanks and thus reduce the manufacturing footprint, or in general to a. reduce the amount of feed added throughout a fed-batch (FB) process, or b. reduce CSPR by using concentrated medium in continuous processes such as perfusion cell culture, thereby potentially increasing the volumetric titer.

[0044] Another important property of amino acid derivatives is that they must be readily available to cells to support metabolic requirements, especially when used in media for perfusion applications. Many amino acid derivatives are not readily bioavailable and require the release of enzymes to cleave them, thereby releasing the bioavailable, authentic amino acid. As a result, many highly soluble amino acid derivatives may not be suitable for cell culture media. Because transport and storage of liquid media are more complicated, cell culture media are typically produced as dry powder media that are dissolved in an appropriate amount of aqueous liquid, such as water or an aqueous buffer, before use.

[0045] Simple dissolution of powdered cell culture media is often complicated by substances, particularly amino acids, that are poorly soluble in aqueous liquids. For example, L-tyrosine has a solubility of 0.4 g / L in water at 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. It has been found that N-norvalyl-L-tyrosine, on the one hand, has a higher solubility in aqueous solutions than tyrosine, and on the other hand, can be used as a partial or preferably complete substitute for tyrosine and is equally suitable as a cell culture medium component.

[0046] The cell culture medium according to the present invention may contain native tyrosine and its salts, as well as N-norvalyl-L-tyrosine and its salts. When the medium is a feed medium or another medium additive added to a basal medium containing native tyrosine or its salts, said feed medium or medium additive preferably contains only N-norvalyl-L-tyrosine or its salts, and does not contain native tyrosine or its salts. N-norvalyl-L-tyrosine or a salt thereof is equally suitable as native tyrosine as a tyrosine source and can therefore be used as the sole tyrosine source or in mixtures with other tyrosine derivatives or native tyrosine or a salt thereof, where N-norvalyl-L-tyrosine or a salt thereof can be used as a complete substitute that is similarly bioavailable and has a higher availability, eliminating the need to add native tyrosine or a salt thereof to any cell culture medium containing N-norvalyl-L-tyrosine or a salt thereof.

[0047] The overall concentration of N-norvalyl-L-tyrosine in the ready-to-use liquid basal / perfusion medium and in the feed medium or medium supplement is highly flexible. The upper limit is defined only by the solubility of N-norvalyl-L-tyrosine in the respective medium. As a result, it is typically possible to produce liquid media with N-norvalyl-L-tyrosine concentrations of up to 100 mmol / L or more, e.g., 0.1 to 100 mmol / L, preferably 1 to 60 mmol / L. To further enhance the solubility of N-norvalyl-L-tyrosine, salts can be formed by reacting N-norvalyl-L-tyrosine with a suitable acid and / or base, with the hydrochloride salt being preferred.

[0048] The powdered cell culture medium of the present invention is preferably produced by mixing all components and grinding them. Mixing the components is well known to those skilled in the art of producing dry powdered cell culture media by grinding. Mixing the components is well known to those skilled in the art of producing dry powdered cell culture media by grinding. Preferably, all components are thoroughly mixed in a dry state so that all parts of the mixture have nearly the same composition. For homogeneous cell growth, the more uniform the composition, the better the quality of the resulting medium. The grinding can be carried out by any type of mill suitable for producing powdered cell culture media. Typical examples are a ball mill, a pin mill, a Fitz mill, or a jet mill. Preferred are pin mills, Fitz mills, or jet mills, and most preferred are pin mills.

[0049] Those skilled in the art know how to implement such a mill. Large scale equipment mills with disc diameters of about 40 cm, for example pin mills, typically operate at 1 to 6500 revolutions per minute, preferably 1 to 3000 revolutions per minute. Milling can be carried out under standard milling conditions and results in a powder with a particle size of 10 to 300 μm, most preferably 25 to 120 μm. Preferably, all components of the mixture subjected to milling are dry, meaning that if they contain water, they contain only 10% or less, preferably 5% or less, and most preferably 2% or less of water crystals by weight of unbound or ungrouped water molecules. In a preferred embodiment, the grinding is carried out in an inert atmosphere, the preferred inert protective gas being nitrogen.

[0050] In another preferred embodiment, all components of the mixture are frozen prior to grinding. Freezing of the components prior to grinding can be done by any means that ensures cooling of the components to a temperature below 0°C, and most preferably below -20°C. In a preferred embodiment, freezing is done by liquid nitrogen. This means that the components are treated with liquid nitrogen, for example, by pouring liquid nitrogen into the container in which they are stored prior to introduction into the mill. In a preferred embodiment, the container is a feeder. If the container is a feeder, preferably, the liquid nitrogen is introduced near or adjacent to the feeder where the components are introduced. Typically, the components are treated with liquid nitrogen for 2 to 20 seconds. Preferably, the cooling of the ingredients is done in such a way that all ingredients entering the mill are at a temperature below 0°C, most preferably below -20°C.

[0051] In a preferred embodiment, all ingredients are placed in a container from which the mixture is transferred into a feeder, most preferably a metering screw feeder. In the feeder, the ingredients are sometimes further mixed and additionally cooled, depending on the type of feeder. The cooled mixture is then transferred from the feeder to a mill so that the mixture to be ground in the mill still has a temperature preferably below 0°C, more preferably below -20°C. Typically, the mixing time, which means the residence time of the mixture of ingredients in the feeder, is greater than 1 minute, preferably 15 to 60 minutes. Metering screw feeders, also known as dose snails, typically run at speeds of 10 to 200 revolutions per minute, preferably 40 to 60 revolutions per minute. Typically, the temperature of the mill is maintained at -50 to +30°C. In a preferred embodiment, the temperature is maintained at about 10°C. The oxygen level during milling is preferably less than 10% (v / v).

[0052] The process can be carried out, for example, batchwise or continuously. In a preferred embodiment, the process according to the invention is made continuous by permanently charging a mixture of ingredients into a feeder for cooling over a predetermined period of time, and then permanently charging the cooled mixture from the feeder into a mill. After grinding, the resulting dry powder medium may be further compressed to increase particle size, for example by dry pressing in a roll press.

[0053] For use of a powdered medium, an aqueous liquid, preferably water (most particularly distilled and / or deionized water or purified water or water for injection) or an aqueous buffer solution, is added to the medium and the components are mixed until the medium is completely dissolved in the solvent, producing a ready-to-use liquid medium. The aqueous liquid may also contain saline, soluble acid or base ions to 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. It is also possible to add further substances to the mixture of cell culture medium and solvent, such as buffer substances for adjusting the pH, fetal calf serum, sugars, etc. The resulting liquid cell culture medium is then contacted with the cells to be grown or maintained.

[0054] While medium compositions containing high concentrations of tyrosine exhibit turbidity when mixed with aqueous liquids due to undissolved tyrosine, cell culture media according to the present invention in which tyrosine is partially or preferably entirely replaced with N-norvalyl-L-tyrosine provide clear solutions, as shown by the turbidity measurements in Example 2 and FIG. 2. In addition, liquid media containing N-norvalyl-L-tyrosine instead of tyrosine have been found to be more stable when stored for extended periods of time. Liquid media containing tyrosine show a decrease in tyrosine concentration after about 20 days, while media containing N-norvalyl-L-tyrosine are stable for more than 50 days, typically more than 70 days. Preferably, the media are stored at room temperature or below, e.g., 4°C to RT, protected from light. Thus, the present invention is also directed to liquid cell culture media containing N-norvalyl-L-tyrosine but not containing tyrosine, that are stable and do not exhibit degradation of N-norvalyl-L-tyrosine for 50 days, preferably 70 days.

[0055] The present invention further comprises: a) providing a bioreactor; b) mixing the cells to be cultured with the cell culture medium according to the invention; c) incubating the mixture of step b). The present invention is directed to a process for culturing cells according to the present invention. Optionally, the liquid cell culture medium is produced by dissolving a dry powder medium containing N-norvalyl-L-tyrosine in an aqueous liquid.

[0056] In one embodiment, the bioreactor is a perfusion bioreactor. A bioreactor is any vessel or tank in which cells can be cultured. Incubation is typically under appropriate conditions, such as an appropriate temperature. Those skilled in the art are aware of appropriate incubation conditions to support or maintain cell growth / culture. A perfusion bioreactor is a bioreactor in which perfusion cell culture can be performed. It typically includes a bioreactor vessel that is closed during cell culture, an agitator 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 in the harvest line that retains the cells while collecting the liquid portion of the harvest. A review of 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.

[0057] Thus, the present invention also provides a process for perfusion cell culture comprising culturing cells in a perfusion bioreactor equipped with a medium inlet and a harvest outlet, wherein i. during the cell culture process, continuously or once or several times, a cell culture medium containing N-norvalyl-L-tyrosine is introduced into the bioreactor through the medium inlet; ii. During the cell culture process, either continuously or once or several times, the harvest is removed from the bioreactor via the harvest outlet and directed to said process. The cell culture medium added in step i. can have a base concentration equivalent to the cell culture medium in the bioreactor, which can be concentrated.

[0058] Additionally, in one embodiment, a second cell culture medium, preferably an enriched medium containing fewer components than the medium in the bioreactor and the medium inserted in step i., is added to the bioreactor via a medium inlet or an additional inlet once or several times during the cell culture process, wherein the second cell culture medium contains N-norvalyl-L-tyrosine. Preferably, the addition of the second cell culture medium is performed without increasing the overall VVD for at least 50% of the time, preferably at least 75% of the time, during the perfusion phase. This means that when the second cell culture medium is inserted into the bioreactor at a specific VVD, the VVD of the basal medium is preferably reduced by at least the VVD of the second medium.

[0059] The performance of cell culture perfusion processes is known to those skilled in the art. Typically, this is done by inoculating a bioreactor with basal medium and cells. The inoculation cell density is typically 0.5-10 million cells / mL, preferably 0.5-1.0 million cells / mL. It is possible to run the process in perfusion mode directly from the beginning, i.e., immediately after seeding the cells. However, the process is preferably first run in batch mode for a period during which the cell count (VCD) increases. Typically, the process is run in batch mode for 2-8 days, preferably 3-5 days, after which perfusion is initiated. In any case, perfusion is preferably turned on before exponential growth ceases. This can be assessed, for example, in previous cell line characterization experiments.

[0060] Typically, perfusion is then turned on by setting a constant perfusion rate and a constant bleed rate to reach a constant VCD. However, perfusion cell culture can also be performed as dynamic perfusion, where the VCD is not constant. Those skilled in the art are aware of various ways to perform perfusion cell culture. The present invention has been found to be highly suitable for the preparation of feed media. Due to limited availability of certain amino acids, especially at the concentrations required for the feed media, feed media could not be prepared at the desired high concentrations or they had to be prepared under drastic pH conditions, such as a very basic pH. This could adversely affect the supply of nutrients to the cells and, to some extent, promote cell death due to exposure to extreme basic pH values. As a result, there is a need for a feed medium that contains all the required components at high concentrations in one feed. Additionally, the pH of the feed should not adversely affect the cell culture.

[0061] N-norvalyl-L-tyrosine has improved solubility, can be used in highly concentrated feeds, and has been found to have no adverse, and sometimes even beneficial, effects on cell growth and / or productivity at pH below 8.5. The present invention is therefore also directed to a feed medium either in the form of a powdered medium or after dissolution in the form of a liquid medium. The resulting liquid medium typically contains N-norvalyl-L-tyrosine at a concentration greater than 10 mmol / l, or greater than 60 mmol / l, preferably 10-60 mmol / l, and preferably has a pH of 8.5 or less. In a preferred embodiment, the pH is 6.5 to 7.8. Preferably, the feed medium does not contain tyrosine.

[0062] The present invention also provides - loading the cells and aqueous cell culture medium into a bioreactor; - incubating the cells in a bioreactor; - adding cell culture medium to the bioreactor continuously or once or several times during the cell incubation period; wherein the added cell culture medium preferably has a pH of less than pH 8.5 and contains N-norvalyl-L-tyrosine; The present invention is directed to a process for culturing cells in a bioreactor according to the method of claim 1. Typically, the medium contains 15 to 150 g / L, preferably 25 to 150 g / L, of solid components that dissolve in the aqueous liquid.

[0063] In one embodiment, the medium added is a feed medium and the process is a fed-batch process. In another embodiment, the medium added is a perfusion medium and the process is a perfusion process. It has been discovered that N-norvalyl-L-tyrosine can be used to obtain a feed medium containing all necessary feed components at high concentrations (total concentration of 100-250 g / L). In contrast to known processes in which two or more different feed media are required to feed a bioreactor, the present invention provides a medium and method that allows the use of a single feed medium containing all components at high concentrations. In addition, the pH of the feed medium according to the present invention is typically less than 8.5.

[0064] In a preferred embodiment, in the process of the invention, the feed medium that is added to the bioreactor continuously during incubation or once or several times during said time always has the same composition. Cell culture processes using N-norvalyl-L-tyrosine have been found to have comparable performance to processes using tyrosine. The quality of proteins produced in cell culture processes using N-norvalyl-L-tyrosine is comparable to that of processes using tyrosine. Due to N-norvalyl-L-tyrosine's high solubility, excellent bioavailability, and improved stability in liquid media compared to tyrosine, N-norvalyl-L-tyrosine is not only a perfect substitute for tyrosine in cell culture media, but also offers more flexibility and advantages compared to tyrosine or its salts.

[0065] 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, and corresponding EP23156964.1 filed February 16, 2023, are hereby incorporated by reference. [Example]

[0066] example The following examples illustrate practical applications of the present invention. Example 1: NL-Norvalyl-L-Tyrosine HCl exhibits increased solubility in water compared to the regular amino acid L-tyrosine and its salts The maximum solubility of NL-Norvalyl-L-Tyrosine HCl (NL-NVa-Tyr HCl) was compared to that of the regular amino acid L-Tyrosine (Tyr) and its salts in water at pH 7.3 ± 0.1 at 25 °C by preparing a supersaturated solution. Measurements were performed after a filtration step (0.22 μm) to remove undissolved compounds. The solution was then analyzed by amino acid analysis using ultra-performance liquid chromatography (UPLC). As shown in Figure 1, Tyr showed a maximum solubility of 2.20 ± 0.02 mM. PTyr (phospho-tyrosine) disodium salt showed a maximum solubility of 167.5 ± 0.8 mM at pH 7.3, while NL-NVa-Tyr HCl showed a maximum solubility of 70.7 ± 0.6 mM.

[0067] Example 2: NL-Norvalyl-L-Tyrosine HCl exhibits increased solubility in 6-fold concentrated CCM compared to the regular amino acid L-tyrosine and its salts The solubility of Tyr 2Na salt 2H2O was compared with that of each derivative or its salt in 6-fold concentrated CCM (modified EX-CELL® Advanced HD Perfusion Medium) at pH 7.3±0.1 and 25°C by turbidity measurements. As shown in Figure 2, the turbidity of NL-NVa-Tyr HCl was much lower compared to that of Tyr 2Na salt 2H2O in 6x concentrated CCM at neutral pH. For NL-NVa-Tyr HCl, the turbidity reduction was greater than 95%. Overall, these results indicate that NL-norvalyl-L-tyrosine and its salts are suitable alternatives for increasing the solubility of cell culture media and feed formulations by replacing L-tyrosine disodium salt dihydrate.

[0068] Example 3: NL-Norvalyl-L-Tyrosine HCl has increased solubility in CCM compared to its respective amino acid, L-Tyrosine, and its salts The maximum solubility of Tyr 2Na salt 2H2O was compared with that of its respective derivative or salt in 6-fold concentrated CCM (modified EX-CELL® Advanced HD Perfusion Medium) at pH 7.3 ± 0.1 and 25 °C by preparing supersaturated derivative solutions. Samples were taken after 30 min of stirring. Measurements were performed after a filtration step (0.22 μm) to remove undissolved compounds. The concentrations of Tyr 2Na salt 2H2O and NL-NVa-Tyr HCl were determined by amino acid analysis using UPLC. The maximum solubility of Tyr 2Na salt 2H2O in the Tyr-depleted modified EX-CELL® Advanced HD Perfusion Medium was found to be approximately 3 mM, while a maximum solubility of approximately 54 mM was detected for NL-Norvalyl-Tyr HCl (Figure 3). This indicates that NL-Norvalyl-Tyr HCl is at least 18-fold more soluble than Tyr 2Na salt 2H2O in the Tyr-depleted modified X-CELL® Advanced HD Perfusion Medium.

[0069] Example 4: A 6x concentrated CCM containing NL-norvalyl-L-tyrosine HCl has increased stability compared to CCM containing L-tyrosine and its salts. It is stable at 4°C and moderately stable at RT, protected from light, for a period of 3 months. Stability studies were performed to monitor the stability of media containing Tyr 2Na salt 2H2O and its corresponding derivative, NL-NVa-Tyr, or its salts, at 4°C and room temperature. Therefore, a 6x concentrated CCM (Tyr-depleted modified EX-CELL® Advanced HD Perfusion Medium) at pH 7.3 ± 0.1 was prepared with 6x concentrations of Tyr 2Na salt 2H2O and NL-NVa-Tyr HCl and stored protected from light for 3 months. The concentrations of Tyr, NL-NVa-Tyr HCl, and NVa were determined by amino acid analysis using UPLC. A filtration step (0.22 μm pore size) was performed prior to amino acid analysis to remove undissolved compounds.

[0070] A modified EX-CELL® Advanced HD Perfusion Medium supplemented with Tyr 2Na salt 2H2O and depleted of Tyr was found to be unstable at 4°C and RT, while the concentration of NL-NVa-Tyr HCl remained constant for 92 days at 4°C. Neither free Tyr nor NVa was detected. At RT, CCM containing NL-NVa-Tyr HCl was found to be stable for 78 days, as shown in Figure 4. Thereafter, the concentration of NL-NVa-Tyr HCl decreased slightly by 10% upon storage at room temperature for more than 3 months. However, neither free Tyr nor NVa was detected. This indicates that the 6x concentrated CCM containing NL-NVa-Tyr HCl is stable when stored protected from light at 4°C and RT for 3 months. The 6x concentrated CCM containing Tyr 2Na salt 2H2O was stable for 6 days at 4°C and 14 days at RT.

[0071] Example 5: NL-Norvalyl-L-Tyrosine HCl can replace its respective amino acid, Tyr2Na2H2O. Cell culture results of IgG1-producing CHOK1 GS clone A. For cell culture experiments, the CHOK1 GS suspension cell line expressing human IgG1 was used. Cells were cultured at 0.3x10 in a starting culture volume of 30 mL. 6 Cells were cultured in triplicate or quadruplicate in modified EX-CELL® Advanced HD Perfusion Medium (Merck, Darmstadt, Germany) using 50 mL spin tubes at a seeding density of 1000 viable cells / mL. Incubations were performed at 37°C, 5% CO2, 80% humidity, and 320 rpm agitation. Tyr derivatives were added to the CCM (Tyr-depleted modified EX-CELL® Advanced HD Perfusion Medium) in place of the amino acid Tyr. The pH of the CCM was neutral (pH 7.3 ± 0.1). The positive control contained Tyr 2Na salt 2H2O, while the negative control contained pTyr 2Na salt, a non-bioavailable tyrosine derivative. Additionally, Pro-Tyr, Gly-Tyr, and Tyr-His were also tested in comparison with NL-NVa-Tyr HCl. Experiments for Gly-Tyr and NL-NVa-Tyr HCl were repeated at least three times, as were positive and negative controls. Viable cell density (VCD = viable cells / mL) and viability were assessed using a Vi-CELL XR (Beckman Coulter, Fullerton, CA). Metabolite concentrations were monitored spectrophotometrically and turbidimetrically using a Cedex Bio HT (Roche Diagnostics, Mannheim, Germany). Amino acid quantification was performed by UPLC after derivatization using the AccQ·TagUltra® reagent kit. Derivatization, chromatography, and data analysis were performed according to the supplier's recommendations (Waters, Milford, MA).

[0072] Figure 5 shows the cell performance of CHOK1 GS clone A in a 7-day batch process. VCD and IgG concentrations were comparable when NL-NVa-Tyr HCl was added to the Tyr-depleted modified EX-CELL® Advanced HD Perfusion Medium compared to the positive control Tyr 2Na Salt 2H2O (Figure 5). Both conditions resulted in a cell viability of 12 ± 1 x 10 cells after 6 days of culture. 6 A maximum VCD of 160 mg / L of viable cells / mL was reached (Figure 5A), resulting in a titer of approximately 160 mg / L after 7 days of culture. Viability was consistently greater than 90% for NLN-NVA-TYR HCl (Figure 5B). A 45% decrease in VCD and greater than 60% decrease in titer were observed for Pro-Tyr compared to the positive control, Tyr 2Na salt 2H2O (Figure 5C). The negative control, pTyr 2Na salt, yielded a titer of 3.8 ± 0.5 × 10 after 7 days of culture. 6 and a maximum IgG concentration of 34 ± 3 mg / L. Overall, Gly-Tyr and Tyr-His showed poorer cellular performance compared to the negative control, pTyr 2Na salt. The concentrations of free Tyr and TyrDer were determined in the spent medium (Figure 5D+E). Because the formation of free Tyr from NL-NVa-Tyr HCl also releases norvaline (NVa), this amino acid was monitored in the supernatant over the course of the batch process (Figure 5F). In conditions where Tyr 2Na salt 2H2O was replaced with NL-NVa-Tyr HCl, the concentration of NL-NVa-Tyr HCl decreased by more than 97% from day 3 to day 6 (Figure 5D). The concentrations of free Tyr (Figure 5E) and NVa (Figure 5F) increased, while the concentration of NL-NVa-Tyr HCl decreased accordingly.

[0073] Batch experiments using various CHO cell lines The applicability of NL-NVa-yr HCl for various bioprocesses was demonstrated by performing batch experiments with various CHO cell lines (1xCHOK1 non-GS, 5xCHOZN GS) producing IgG1 or fusion proteins. Therefore, cells were cultured at 0.3x10 in a starting culture volume of 30 mL. 6 The cells were cultured in triplicate or quadruplicate in modified EX-CELL® Advanced HD Perfusion Medium (Merck Darmstadt, Germany) using 50 mL spin tubes at a seeding density of 1000 viable cells / mL. Incubation was performed at 37°C, 5% CO2, 80% humidity, and 320 rpm agitation. Tyr derivatives were added to CCM (Tyr-depleted modified EX-CELL® Advanced HD Perfusion Medium) in place of the canonical amino acid Tyr. The pH of the CCM was neutral (pH 7.3 ± 0.1). The positive control contained Tyr 2Na salt 2H2O, while the negative control contained pTyr2Na salt. No negative control was used for CHOK1 non-GS clones.

[0074] Viable cell density (VCD = viable cells / mL) and viability were assessed using a Vi-CELL XR (Beckman Coulter, Fullerton, CA). Metabolite concentrations were monitored using a Cedex Bio HT (Roche Diagnostics, Mannheim, Germany) spectrophotometric and turbidimetric assay. Amino acid quantification was performed by UPLC after derivatization using the AccQ·TagUltra® reagent kit. Derivatization, chromatography, and data analysis were performed according to the supplier's recommendations (Waters, Milford, MA).

[0075] Example 6: Confirmation of NL-Norvalyl-Tyr HCl performance with CHOK1 non-GS producing IgG1. Figure 6 shows the cell performance of the CHOK1 non-GS clone producing IgG1 in a batch experiment. Overall, the results show similar cell performance for NL-NVa-Tyr HCl compared to the positive control, Tyr 2Na salt 2H2O (Figure 6A+B). Both conditions yielded 15-16 x 10 cells after 7 days of culture. 6The maximum VCD in the range of viable cells / mL was reached, and viability was always above 95%. NL-NVa-Tyr HCl resulted in a slightly increased titer production of 23% (Figure 6C). After 6 days of culture, the NL-NVa-Tyr HCl concentration of 1513 μM was completely depleted (Figure 6D), while the Tyr concentration increased to a maximum of 783.5 μM on day 5 and then decreased, similar to the positive control (Figure 6E).

[0076] Example 7: Confirmation of NL-Norvalyl-Tyr HCl performance with CHOZN GS clone A producing fusion protein. Results for CHOZN GS clone A, which produces the fusion protein, show similar cell performance in NL-NVa-Tyr HCl compared to the positive control, Tyr 2Na 2H2O (Figure 7A-C). Both conditions yielded approximately 11.0 x 10 cells by day 7 of culture. 6 A maximum VCD of viable cells / mL and titer production in the range of 230 mg / L were achieved. Viability was consistently above 97%. After 5 days of culture, NL-NVa-Tyr HCl was completely depleted (Figure 7D), while the concentration of free Tyr increased from 0 μM on day 0 to a maximum of 1080 μM on day 4, then decreased similarly to the positive control (Figure 7E). In contrast, the negative control, pTyr 2Na salt, maintained its initial concentration, and free Tyr was not detected.

[0077] Example 8: Confirmation of NL-Norvalyl-Tyr HCl performance with CHOZN GS clone B producing fusion protein. Similar to CHOZN GS clone A, CHOZN GS clone B, which produces the fusion protein, also exhibits similar cell performance with NL-NVa-Tyr HCL as well as the positive control (Figure 8A-C). For both conditions, on day 7 of culture, the maximum VCD was 9-10x10. 6 Viability was consistently above 97% in both conditions (Figure 8B). After 4 days of culture, NL-NVa-Tyr HCl decreased by more than 90% (Fig. 8D), while the concentration of free Tyr increased from 0 μM on day 0 to a maximum of 1187 μM on day 4 and then decreased similarly to the positive control (Fig. 8E). In contrast, the negative control, pTyr 2Na salt, maintained its initial concentration, and little or no free Tyr was detected.

[0078] Example 9: Confirmation of NL-Norvalyl-Tyr HCl performance with CHOZN GS clone C producing IgG1. Figure 9 shows the cell performance of CHOZN GS clone C producing IgG1 in a batch experiment. Overall results show similar cell performance for NL-NVa-Tyr HCl compared to the positive control, Tyr 2Na salt 2H2O (Figure 9A-C). Both conditions yielded 6.5-8 x 10 cells after 7 days of culture. 6 Maximum VCD in the range of viable cells / mL and titer production in the range of 430 mg / L were reached. Viability was consistently above 95% until day 6. The NL-NVa-Tyr HCl concentration of 1513 μM was completely depleted after 4 days of culture (Figure 9D), while the Tyr concentration increased to a maximum of 1017 μM on day 4 and then decreased, similar to the positive control (Figure 9E).

[0079] Example 10: Confirmation of NL-Norvalyl-Tyr HCl performance with CHOZN GS clone D producing IgG1. Results for IgG1-producing CHOZN GS clone D show similar cell performance with NL-NVa-Tyr HCl compared to the positive control, Tyr 2Na salt 2H2O (Figure 10A-C). The positive control showed 20 ± 1 x 10 6 The maximum VCD reached 16.9 ± 0.5 × 10 viable cells / mL and a titer of 668 ± 55 mg / L. NL-NVa-Tyr HCl reached 16.9 ± 0.5 × 10 viable cells / mL after 6 days of culture. 6 and resulted in a maximum VCD of 609 ± 39 mg / L after 7 days. Viability remained consistently above 95% by day 6. After 5 days of culture, NL-NVa-Tyr HCl decreased by more than 97% (Figure 10D), while the concentration of free Tyr increased from 0 μM on day 0 to a maximum of 968 ± 133 μM on day 5 and then decreased similarly to the positive control (Figure 10E). The concentration of NVa increased to a maximum of 1590 μM on day 5 and remained in this range thereafter (Figure 10F). In contrast, the negative control, pTyr 2Na salt, maintained its initial concentration, and free Tyr was not detected.

[0080] Example 11: Confirmation of NL-Norvalyl-Tyr HCl performance with CHOZN GS clone E producing IgG1. Figure 11 shows the cell performance of CHOZN GS clone E producing IgG1 in a batch experiment. Overall results show similar cell performance for NL-NVa-Tyr HCl compared to the positive control, Tyr 2Na salt 2H2O (Figures 11A-C). In both conditions, on day 7 of culture, 16x10 6 The maximum VCD in the range of viable cells / mL was reached. The positive control resulted in a titer of 685 ± 5 mg / L, and NL-NVa-Tyr HCl reached a titer of 597 ± 8 mg / L after 7 days of culture. Viability was consistently >98%. After 5 days of culture, the 1513 μM NL-NVa-TyrHCl concentration was completely depleted (Fig. 11D), while the free Tyr concentration increased to a maximum of 911 ± 120 μM on day 5 and then decreased similarly to the positive control (Fig. 11E). The increase in NVa was inversely proportional to the decrease in NL-NVa-TyrHCl up to an NVa concentration of 1300 μM, which was largely maintained from day 5 onwards (Fig. 11F). In contrast, the negative control reduced VCD by approximately 2.0 × 10 6 The initial concentration was maintained at 100 mg / mL, reaching a maximum titer of less than 100 mg / L, and no free Tyr was detected.

[0081] Example 12: Antibody quality The quality of antibody produced in a control batch process using CHOK1 GS clone A and CHOZN GS clone D (CCM containing Tyr 2Na salt 2H2O) was compared with the quality of antibody produced in CCM depleted of Tyr and supplemented with NL-NVa-Tyr HCl. Antibodies were purified from cell culture supernatants using Protein A PhyTips® (PhyNexus Inc, San Jose, CA). 15 μg of purified protein was subjected to released N-glycan analysis. Samples were prepared according to the instructions in the GlycoWorks™ RapiFluor-MS™ N-Glycan Kit. After sample preparation, glycans were stored at 4°C in the autosampler and separated on an ACQUITY UPLC system using a Glycan BEH Amide column (2,1 x 150 mm, Waters Art-Nr. 186004742) heated to 45°C. The injection volume was 18 μL. Separation was performed according to the following gradient of eluent A (50 mM ammonium formate pH 4.4) and eluent B (100% ACN):

[0082] [Table 1]

[0083] Glycan species were quantified using fluorescence detection at 425 nm after excitation at 265 nm. Glycans were identified by mass using a Synapt G2 HDMS. The results obtained for glycosylation (Figure 12) show that there is no difference between the control condition, Tyr 2Na salt 2H2O, and the condition in which Tyr was exchanged with NL-NVa-Tyr HCl. Thus, the amino acid exchange does not affect the glycosylation pattern of the IgG produced in this study. Antibody aggregation was measured using size exclusion chromatography on a Water Acquity UPLC system with 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. Sample concentrations were adjusted to 1.0 mg / mL using storage buffer after IgG purification, and detection was performed using absorbance at 214 nm.

[0084] The results obtained for aggregation (Figure 13) show that there is no difference between the control condition, Tyr 2Na salt 2H2O, and the condition in which Tyr was exchanged with NL-NVa-Tyr HCl. Thus, the amino acid exchange does not affect the aggregation of the IgG produced in this study. Antibody fragmentation was measured by capillary electrophoresis using CE-SD on a CESI 8000 (Beckman Coulter / Sciex) according to the manufacturer's instructions. 100 μg of purified antibody sample was used in a total volume of 45 μL of storage buffer. Samples were not alkylated. Prior to measurement, samples were mixed with an internal standard (10 kDa) and SDS sample buffer, followed by an incubation step of 70°C and 500 rpm for 5 minutes for IgG produced by CHOK1 GS clone A or 60°C and 500 rpm for 10 minutes for IgG produced by CHOZN GS clone D.

[0085] The fragmentation results (Figure 14) show no difference between the control condition, Tyr 2Na Salt 2H2O, and the condition where Tyr was exchanged with NL-NVa-Tyr HCl. Thus, the amino acid exchange does not affect the fragmentation of the IgG produced in this study. Apostol et al. reported that norvaline can be incorporated in place of leucine in the α and β subunits of recombinant human hemoglobin expressed in Escherichia coli (Apostol, I., Levine, J., Lippincott, J., Leach, J., Hess, E., Glascock, C.B., Weickert, M.J., & Blackmore, R. (1997)). Norvaline incorporation at the leucine position of recombinant human hemoglobin expressed in Escherichia coli. Journal of Biological Chemistry, 272(46), 28980-28988. https: / / doi.org / 10.1074 / jbc.272.46.28980). Norvaline exhibits a charge equivalent to leucine but is smaller and less hydrophobic. The possibility of incorporation of N-Va instead of Leu can be investigated by intact mass spectrometry and peptide mapping.

[0086] Middle-up analysis of recombinant mAb after reduction The molecular weights of the light and heavy chains of the mAb obtained after reduction were measured using UHPLC (Vanquish Horizon UHPLC, Thermo Fisher Scientific) coupled with an ESI-Q-ToF mass spectrometer (Impact II, Bruker Daltonics). Briefly, 500 ng of sample was loaded onto a reversed-phase column (1000 Å, 5 μm, 2.1 x 50 mm, Agilent) thermostated at 80 °C at a flow rate of 0.6 mL / min and eluted with the gradient shown in Table 2. LC-MS gradients were used for intact mass analysis. MS analysis was performed using an Impact II mass spectrometer equipped with an ESI source (Bruker Daltonics). MS acquisition was performed in positive mode with the endplate offset and capillary voltage set to 500 V and 4500 V, respectively. The nebulizer and drying gas were set to 3.0 bar and 12.0 L / min, respectively. MS spectra were acquired at a scan rate of 1 Hz over the m / z range of 300–3000. Calibration was performed using an internal lock mass of 1221.9906. Charge state deconvolution was performed using a maximum entropy algorithm.

[0087] [Table 2]

[0088] Peptide mapping - Trypsin digestion Briefly, 10 μL of recombinant mAb (diluted at 1 mg / mL in 50 mM ammonium bicarbonate) was denatured and reduced by adding 1 μL of 1% Protease Max and 1 μL of 100 mM dithiothreitol, followed by incubation at 56°C for 30 min. Free cysteines were then alkylated by adding 1 μL of 200 mM iodoacetamide, followed by incubation at RT for 45 min in the dark. Digestion was then performed by adding 35 μL of 50 mM ammonium bicarbonate and 1 μL of trypsin solution prepared at 1 μg / μL (incubated overnight at 37°C). Trypsin digestion was stopped by adding 1 μL of 100% formic acid. Finally, the sample was centrifuged at 13,000 rpm for 10 min, and the supernatant was transferred to an LC-MS vial prior to analysis.

[0089] - LC-MS / MS experiments The tryptic digest was subjected to RP-HPLC-UV-ESI-MS and MS / MS analysis using a Vanquish Horizon UHPLC (Thermo Fisher Scientific) coupled to an Impact II mass spectrometer (Bruker Daltonics). The tryptic digest (1 μg) was loaded onto an ACQUITY UPLC CSH C18 column (1.7 μm, 2.1 × 150 mm, Waters) thermostated at 60 °C at a flow rate of 0.4 mL / min and eluted with the gradient shown in Table 3.

[0090] [Table 3]

[0091] MS analysis was performed using an Impact II mass spectrometer equipped with an ESI source (Bruker Daltonics). MS acquisition was performed in positive mode with the endplate offset and capillary voltage set to 500 and 4500 V, respectively. The nebulizer and drying gas were set to 0.4 bar and 4.0 L / min (180 °C), respectively. MS spectra were acquired at a scan rate of 2 Hz over the m / z range of 50–2200. Acquisition was performed using the Intensity Dependent Acquisition Speed ​​(IDAS) mode with a cycle time of 2 s. Calibration was performed using an internal lock mass of 1221.9906. Peptide identification was performed using PEAKS XPro software (BSI informatics). Incorporation of L-norvaline in place of leucine was not detected.

[0092] Example 13: Small-scale dynamic perfusion experiment Figure 15 shows the cell performance of CHOZN GS clone D producing IgG1 in a dynamic perfusion experiment. Overall results show similar cell performance for NL-NVa-Tyr HCl compared to the positive control, Tyr 2Na salt 2H2O (Figure 15A-C). Both conditions yielded 100x10 6 The maximum VCD was reached in the range of viable cells / mL. The positive control yielded a maximum titer of 1425 ± 39 mg / L, and NL-NVa-Tyr HCl reached a titer of 1400 ± 22 mg / L after 9 days of culture. Viability was consistently above 90% until day 5. From day 6 onwards, viability decreased slightly and remained between 85 and 90%. After 1 day of culture, the NL-NVa-Tyr HCl concentration of 1513 μM was completely depleted (Figure 15D), while the concentration of free Tyr increased to a maximum of 1106 ± 52 μM on day 1 and then decreased similarly to the positive control (Figure 15E). The NVa concentration was maintained at approximately 1000 μM throughout the process. The negative control was approximately 13 × 10 6The VCD of viable cells / mL was maintained, reaching a maximum titer of 253 ± 3 mg / L on day 6, the initial concentration was maintained, and no free Tyr was detected.

[0093] Figure 16 shows the cell performance of CHOZN GS clone F producing IgG1 in a dynamic perfusion experiment. Overall results show similar cell performance for NL-NVa-Tyr HCl compared to the positive control, Tyr 2Na salt 2H2O (Figures 16A-C). Both conditions showed a 70x10 6 The maximum VCD in the range of viable cells / mL was reached. The positive control yielded a maximum titer of 1335 ± 38 mg / L, and NL-NVa-Tyr HCl reached a titer of 1379 ± 23 mg / L after 9 days of culture. Viability was consistently above 95% until day 6, at which point viability decreased to below 90%. After 1 day of culture, the concentration of 1513 μM NL-NVa-Tyr HCl was completely depleted (Figure 16D), while the concentration of free Tyr increased to a maximum of 1143 μM on day 1 and then decreased, similar to the positive control (Figure 16E).

[0094] Figure 17 shows the cell performance of CHO DG44 producing IgG1 in a dynamic perfusion experiment. Overall results show similar cell performance for NL-NVa-Tyr HCl compared to the positive control, Tyr 2Na salt 2H2O (Figures 17A-C). Both conditions yielded 80x10 cells at day 6. 6 The maximum VCD in the range of viable cells / mL was reached. The positive control yielded a maximum titer of 1304 ± 18 mg / L, and NL-NVa-Tyr HCl reached a titer of 1360 ± 3 mg / L after 7 days of culture. Viability was consistently above 90% until day 7. Thereafter, viability fell below 90%. After 1 day of culture, the concentration of NL-NVa-Tyr HCl, 1513 μM, was completely depleted (Figure 17D), while the concentration of free Tyr increased to a maximum of 1384 ± 470 μM on day 2 and then decreased, similar to the positive control (Figure 17E).

[0095] Figure 18 shows the cell performance of CHOZN GS clone G producing IgG1 in a dynamic perfusion experiment. Overall results show similar cell performance for NL-NVa-Tyr HCl compared to the positive control, Tyr 2Na salt 2H2O (Figures 18A-C). Both conditions yielded 100x10 6 The maximum VCD was reached in the range of viable cells / mL. The positive control yielded a maximum titer of 1425 ± 39 mg / L, and NL-NVa-Tyr HCl reached a titer of 14,000 ± 22 mg / L after 9 days of culture. Viability was consistently above 90% until day 5. From day 6 onwards, viability decreased slightly and remained at 85-90%. After 1 day of culture, the concentration of 1513 μM NL-NVa-Tyr HCl was completely depleted (Figure 18D), while the concentration of free Tyr increased to a maximum of 1131 ± 69 μM on day 1 and then decreased, similar to the positive control (Figure 18E).

Claims

1. A cell culture medium containing N-norvalyl-L-tyrosine.

2. 2. The cell culture medium of claim 1, wherein the cell culture medium comprises N-norvalyl-L-tyrosine hydrochloride.

3. 3. The cell culture medium according to claim 1, wherein the medium contains N-norvalyl-L-tyrosine but does not contain tyrosine.

4. 4. The cell culture medium according to claim 1 or 3, characterized in that the cell culture medium is a dry powder medium.

5. The cell culture medium according to any one of claims 1 to 3, characterized in that the cell culture medium is a liquid medium having a pH of 8.5 or less and containing N-norvalyl-L-tyrosine at a concentration of 10 mmol / L to 60 mmol / L.

6. 6. The cell culture medium according to claim 1, wherein the concentration of the cell culture medium is in a concentrated form that is X times greater than the concentration of the medium in use, where X is between 1.5 and 100.

7. The cell culture medium according to any one of claims 1 to 6, characterized in that the liquid medium has a pH of 6.0 to 8.

5.

8. 8. The cell culture medium according to claim 1, characterized in that the cell culture medium comprises 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.

9. a) mixing N-norvalyl-L-tyrosine with other components of the cell culture medium; b) subjecting the mixture of step a) to grinding.

9. A method for producing a cell culture medium according to any one of claims 1 to 8, by

10. 10. The method for producing a cell culture medium according to claim 9, characterized in that step b) is carried out in a pin mill, a Fitz mill or a jet mill.

11. 11. The method for producing a cell culture medium according to claim 9 or 10, characterized in that the mixture from step a) is cooled to a temperature below 0°C prior to grinding.

12. a) Providing a bioreactor b) optionally producing a liquid cell culture medium by dissolving the dry powder medium according to any one of claims 1 to 8 in an aqueous solution. c) Mixing the cells to be cultured with the liquid cell culture medium according to any one of claims 1 to 8. d) incubating the mixture of step c). A process for culturing cells according to.

13. 13. The process according to claim 12, characterized in that the bioreactor is a perfusion bioreactor.

14. The process for culturing cells is Culturing cells in a perfusion bioreactor equipped with a medium inlet and a harvest outlet, i. continuously or once or several times during the cell culture process, fresh liquid cell culture medium containing N-norvalyl-L-tyrosine is introduced into the bioreactor through the medium inlet; ii. The harvest is removed from the bioreactor via the harvest outlet continuously or once or several times during the cell culture process; 14. A process according to claim 12 or 13, characterized in that it is a process for perfusion cell culture.

15. 15. The process according to claim 14, characterized in that during the perfusion phase, cell culture medium containing N-norvalyl-L-tyrosine is continuously inserted into the bioreactor via the medium inlet and harvest is continuously removed from the bioreactor.

16. a) charging the cells and aqueous cell culture medium into a bioreactor; b) incubating the cells in a bioreactor; c) adding the cell culture medium, in this case the feed medium, continuously or once or several times throughout the incubation of the cells in the bioreactor; wherein the feed medium is a liquid cell culture medium containing N-norvalyl-L-tyrosine; A fed-batch process for culturing cells in a bioreactor, according to

17. 17. The fed-batch process of claim 16, characterized in that the liquid feed medium added in step c) is prepared by dissolving the dry powdered cell culture medium of any one of claims 1 to 8.

18. 18. The fed-batch process of claim 16 or 17, wherein the liquid feed medium has a pH of less than 8.5 and contains at least N-norvalyl-L-tyrosine at a concentration greater than 10 mmol / L.

19. 19. The fed-batch process according to any one of claims 16 to 18, characterized in that the liquid feed medium does not contain tyrosine.

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