Cell culture medium

Abstract

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

Description

[0001] The present invention relates to cell culture media comprising 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 fully replacing them with N-lactyl derivatives.

[0002] Cell culture media support and maintain cell growth in an artificial environment.

[0003] Depending on the type of organism whose growth should be supported, cell culture media contain a complex mixture of components (sometimes more than a hundred different components).

[0004] Cell culture media required for the propagation of mammalian, insect or plant cells are generally much more complex than those that support the growth of bacteria and yeast.

[0005] The first cell culture media developed consisted of components of undefined composition, such as plasma, serum, embryo extracts or other biological extracts or peptones of undefined composition. Thus, significant progress has been made in 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 more substances known to those skilled in the art.

[0006] Some cell culture media are provided as sterile aqueous liquids. The disadvantage of liquid cell culture media is their shortened shelf life and the difficulties associated with transportation and storage. Thus, many cell culture media are currently provided as finely ground dry powder mixtures. They are manufactured for the purpose of being dissolved in water and / or aqueous solutions and, in the dissolved state, are often designed, together with other supplements, to provide a substantial nutrient matrix for cells for the growth of said cells and / or the production of biopharmaceuticals from said cells.

[0007] Most biopharmaceutical production platforms are based on fed-batch cell culture protocols. The aim is usually to develop high-titer cell culture processes to meet the growing market demand and reduce manufacturing costs. In addition to using high-performance recombinant cell lines, improvements in cell culture media and process parameters are also required to achieve maximum production potential.

[0008] In a fed-batch process, the basal medium supports initial growth and production, while the feed medium prevents nutrient depletion and maintains the production phase. The medium is selected to accommodate different metabolic requirements during different production stages. Process parameter settings - including feeding strategies and control parameters - define the chemical and physical environment suitable for cell growth and protein production.

[0009] The optimization of the feed medium is a major aspect in the optimization of the fed-batch process.

[0010] 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.

[0011] Amino acids (AAs) are essential components of cell culture media as they are crucial for supporting cell growth. Additionally, AAs are key building blocks of recombinant proteins produced using mammalian cell culture technology. The solubility of AAs is a limiting factor hindering the concentration of cell culture media (CCM) and feed formulations. Such concentration will be necessary for developing next-generation manufacturing platforms. In particular, biomanufacturing processes using in-line dilution require highly concentrated formulations to reduce the volume of CCM that must be stored in tanks (= reduce manufacturing footprint), or generally reduce the volume of feed added throughout a fed-batch (FB) process, and thus potentially increase volumetric titer.

[0012] Therefore, it would be advantageous to find ways to improve the solubility of amino acids.

[0013] Although N-lactyl amino acids are known to be highly resistant to proteolysis (Lorbert SJ et al.: Oligomers and oligomeric segments of alpha-hydroxy carboxylic acids and alpha amino acids. US 2004 / 0048347, 2004), it has unexpectedly been found that amino acids in cell culture media can be replaced by their N-lactyl derivatives or their salts. Besides their use as a source of amino acids, the N-lactyl derivatives exhibit higher solubility compared to their corresponding amino acids and can thus be used in highly concentrated formulations.

[0014] In the food industry, particularly in foods such as soy sauce and meat products, N-lactyl amino acids are known as flavor-active amino acids formed from free amino acids by the action of lactyltransferases in Lactobacillus spp. (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.).

[0015] N-lactyl amino acids have also been described in the context of medicine or human cell models. According to Jansen RS et al., N-lactyl 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). In very recent studies, N-lactyl amino acids have been described as extracellular biomarkers associated with intracellular amino acid concentrations in human cell models. These derivatives have been described as useful biomarkers 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.

[0016] N-lactyl amino acids as a cell culture medium component are unknown.

[0017] Accordingly, the present invention relates to a cell culture medium comprising at least one N-lactyl amino acid. If the term amino acid is used hereinafter, 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 know that free amino acids can be used, or H + can be replaced by metal counterions such as Na + to form salts.

[0018] 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.

[0019] In a preferred embodiment, the cell culture medium comprises one or more components of formula I:

[0020]

[0021] wherein R1 + is H+ or metal ions such as Na + , K + , Mg 2+ , Ca 2+ , Li + , preferably Na + ,

[0022] R2 is a characteristic residue of the amino acid. In the case where the amino acid is leucine, the component of formula I will be

[0023]

[0024] In the case where the amino acid is isoleucine, the component of formula I will be

[0025]

[0026] In a preferred embodiment, the cell culture medium contains the sodium salt of N-lactyl amino acid. This means that, preferably, R1 + is Na + .

[0027] In a preferred embodiment, the cell culture medium is a dry powder medium.

[0028] In one embodiment, especially if 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 natural amino acids. This means it contains, for example, N-lactyl leucine and the corresponding natural leucine. In this case, natural means unmodified amino acids and / or their salts.

[0029] In another preferred embodiment, the cell culture medium is a feeding medium.

[0030] The feeding medium can contain one or more N-lactyl amino acids and the corresponding natural amino acids, but it can also contain only one or more N-lactyl amino acids without the corresponding natural amino acids.

[0031] In a preferred embodiment, if the cell culture medium is a feeding medium, the medium contains one or more N-lactyl amino acids but does not contain the corresponding natural amino acids.

[0032] In another preferred embodiment, the cell culture medium is a liquid medium having a pH of 8.5 or lower and containing at least one N-lactyl amino acid according to formula I at a concentration higher than 10 mmol / l. In the case of a feeding medium, the concentration is usually higher than 30 mmol / l. The upper limit is only limited by the solubility of the lactyl amino acid. Solubility may depend on the solvent, pH and salt concentration. Thus, it is generally possible to produce a liquid medium with a lactyl amino acid concentration of up to 500 mmol / l or more.

[0033] 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.

[0034] 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.

[0035] The present invention further relates to a method for producing a cell culture medium according to the present invention by:

[0036] a) Mixing one or more N-lactyl amino acids according to formula I with the other components of the cell culture medium

[0037] b) Subjecting the mixture of step a) to grinding.

[0038] In a preferred embodiment, step b) is carried out in a pin mill, a Fitz mill or a jet mill.

[0039] In another preferred embodiment, the mixture from step a) is cooled to a temperature below 0 °C before grinding.

[0040] The present invention further relates to a process for culturing cells by:

[0041] a) Providing a bioreactor

[0042] b) Mixing the cells to be cultured with the cell culture medium according to the present invention.

[0043] c) Incubating the mixture of step b).

[0044] In one embodiment, the bioreactor is a perfusion bioreactor.

[0045] The present invention also relates to a fed-batch process for culturing cells in a bioreactor by:

[0046] - Filling the bioreactor with cells and an aqueous cell culture medium

[0047] - Cells incubated in a bioreactor

[0048] - The cell culture medium is added to the bioreactor continuously throughout the entire time or one or more times during the cell incubation time, and in this case, the cell culture medium is a feeding medium, and the feeding medium is a cell culture medium according to the present invention containing at least one N-lactyl amino acid.

[0049] Preferably, the feeding medium has a pH lower than 8.5 and contains at least one N-lactyl amino acid at a concentration higher than 10 mmol / l.

[0050] Preferably, the N-lactyl amino acid is N-lactyl leucine and / or N-lactyl isoleucine. Description of the Drawings

[0051] Figure 1 Shows an exemplary structure of a natural amino acid with its characteristic residues;

[0052] Figure 2 Is the maximum solubility of Ile or Lac-Ile in 4Feed depleted of Ile / Leu;

[0053] Figure 3 Is the maximum solubility of Leu or Lac-Leu in 4Feed depleted of Ile / Leu;

[0054] Figure 4 Is the maximum solubility of 4Feed at neutral pH;

[0055] Figure 5 Is the maximum solubility of 4Feed in which Ile and Leu have been replaced by Lac-Ile and Lac-Leu;

[0056] Figure 6 Is the standard curve of Lac-Leu and Lac-Ile;

[0057] Figure 7 Is the stability of lactyl leucine in Cellvento 4Feed-Ile / Leu;

[0058] Figure 8 Shows the VCD within a 17-day fed-batch process, where Lac-Leu or Lac-Ile replaces Leu and Ile in the feed, respectively;

[0059] Figure 9 Shows the IgG produced within a 17-day fed-batch process, where Lac-Leu or Lac-Ile replaces Leu and Ile in the feed, respectively;

[0060] Figure 10 Shows lactate production during a 17-day fed-batch process, where Lac-Leu or Lac-Ile replace Leu and Ile in the feed, respectively;

[0061] Figure 11 Shows quantification of Leu in the used medium during a 17-day fed-batch process with replacement of Leu and Ile in the feed by Lac-Leu or Lac-Ile, respectively;

[0062] Figure 12 Shows quantification of Ile in the used medium during a 17-day fed-batch process with replacement of Leu and Ile in the feed by Lac-Leu or Lac-Ile, respectively;

[0063] Figure 13 Shows the glycosylation of IgG1 produced in a control process or in a process where the feed depleted of Ile / Leu is supplemented with Lac-Leu or Lac-Ile;

[0064] Figure 14 Shows the aggregation and fragmentation of IgG1 produced in a control process or in a process where the feed depleted of Ile / Leu is supplemented with Lac-Leu or Lac-Ile;

[0065] Figure 15 Shows the charge variants of IgG1 produced in a control process or in a process where the feed depleted of Ile / Leu is supplemented with Lac-Leu or Lac-Ile;

[0066] Figure 16 , 17 and 18 show the performance of the Lac-Leu and Lac-Ile processes compared to a control of the CHODG44 cell line expressing IgG1; and

[0067] Figure 19 and 20 show the relative concentrations of isoleucine ( Figure 19 ) and leucine ( Figure 20 ) in the used medium in the Lac-Leu and Lac-Ile processes (CHODG44 cells) compared to a normal process using unmodified Ile and Leu.

[0068] Regarding Figures 2 to 20 Further details can be found in the examples.

[0069] N-lactyl amino acids are amino acids covalently linked via their amino group to a lactyl residue. The lactyl residue is the chemical moiety CH3CH(OH)CO- of lactic acid.

[0070] The N-lactyl amino acids according to the invention are products obtainable, for example, by chemical or biotechnological synthesis.

[0071] N-lactyl amino acids can be synthesized in vivo and in microorganisms by enzymes such as lactoyltransferases in Lactobacillus species (Zhao CJ, Schieber A, Ganzle MG: Formation of taste-active aminoacids, 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).

[0072] Also described is the synthesis of N-lactyl amino acids using biocatalytic methods in mixtures containing: enzymes (such as enzymes catalyzing peptide bond formation, such as serine proteases, thiol proteases, metalloproteases, esterases or alkaline proteases), α-hydroxycarboxylic acids (or derivatives, such as the corresponding esters, acyl halides, amides, acid anhydrides or ketenes) and α-amino acids (or derivatives, such as the corresponding esters, acyl halides, amides, acid anhydrides or ketenes) (Lorbert SJ et al.: Oligomers and oligomeric segments of alpha-hydroxy carboxylic acids and alpha amino acids. US 2004 / 0048347, 2004).

[0073] Several chemical processes for the synthesis of N-lactyl amino acids have been described. The synthesis of Lac-Phe is described exemplarily in Example 1 according to 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.

[0074] Other synthetic processes have been described in the literature. For example, Lac-Glu has been synthesized from L-lactic acid and L-glutamate esters 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):1842-1850 to predominantly produce the L-form of Lac-AA. Characterization was performed using LC-MS SRM. Similarly, Lac-Val was synthesized from L-lactic acid and H-Val-OBzl tosylate in two steps with an overall yield of 56% after hydrogenolysis. Finally, the synthesis by a single coupling reaction between an amino ester-protected amino acid and (+)-(S)-lactic acid in the presence of one equivalent of (benzotriazol-1-yloxy-tris[pyrollidin-1-yl]- hexafluorophosphate) and an excess of diisopropylethylamine was described in Frerot E, Escher D: Flavored products and a process for their preparation. US5780090, 1998. NMR and LC-MS data for Lac-Glu, Lac-Ala, Lac-Leu, Lac-Ile, N-lactyl tyrosine (Lac-Tyr) and Lac-Met were provided.

[0075] The N-lactyl amino acid is preferably a compound according to formula I

[0076]

[0077] wherein R1 + is H + or a metal ion such as Na + 、K +, Mg 2+ , Ca 2+ , Li + , preferably Na + ,

[0078] R2 is a characteristic residue of an amino acid.

[0079] 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 its characteristic properties so that it 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 with some exemplary characteristic residues R2.

[0080] A cell culture medium according to the present invention is any mixture of components that maintain and / or support the in vitro growth of cells. It may be a complex medium or a chemically defined medium. The cell culture medium can contain all the components required to maintain and / or support the in vitro growth of cells, or only some components, such that further components are added separately. Examples of cell culture media according to the present invention are complete media, which contain all the components required 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. The complete medium is also referred to as a basal medium and typically has a pH of 6.5 to 7.8. The feed medium preferably has a pH below 8.5, preferably 6.0 to 8.5.

[0081] Generally, a cell culture medium according to the present invention is used to maintain and / or support cell growth in a bioreactor.

[0082] A feed or feed medium is a cell culture medium that is not the basal medium that supports the initial growth and production of a cell culture, but is added later to prevent nutrient depletion and maintain the production phase. Compared to the basal medium, the feed medium can have a higher concentration of some components. For example, some components such as nutrients (including amino acids or carbohydrates) can be present in the feed medium at a concentration of about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100x, 200X, 400X, 600X, 800X or even about 1000X the concentration in the basal medium.

[0083] 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, I VERO cells, BHK cells, AK-1 cells, SP2 / 0 cells, L5.1 cells, hybridoma cells or human cells.

[0084] A chemically defined cell culture medium is a cell culture medium that does not contain any substances of undefined chemical composition. 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 other components that may contribute to proteins of less defined chemical composition in the medium. Chemically undefined or less defined chemical components are those components whose chemical composition and structure are unknown, exist in variable composition, or can only be determined with great experimental effort – comparable to the assessment of the chemical composition and structure of proteins such as albumin or casein.

[0085] A powdered cell culture medium or dry powder medium is a cell culture medium that typically results from a grinding process, a freeze-drying process or a dry or wet granulation process. This means that a powdered cell culture medium is a granular particulate medium and not a liquid medium. The term "dry powder" can be used interchangeably with the term "powder"; however, as used herein, "dry powder" only refers to the macroscopic appearance of the particulate material and is not intended to mean that the material is completely free of complexed or agglomerated solvent, unless otherwise stated. Dry powder media resulting from a grinding or freeze-drying process typically have a particle size of less than 0.5 mm, for example 0.05 to 0.5 mm.

[0086] Dry powder media resulting from a dry or wet granulation process, such as by spray drying, wet granulation or dry compaction, typically have a particle size greater than 0.5 mm, for example 0.5 to 5 mm. Dry compaction is typically done in a roll press. US 6,383,810B2 discloses a method for producing an agglomerated eukaryotic cell culture medium powder. The method comprises wetting a dry powder cell culture medium with a solvent and then redrying the wetted medium to obtain a dry agglomerated cell culture medium.

[0087] In one embodiment, the dry powder medium according to the invention is produced by dry compaction.

[0088] The cells to be cultured with the medium according to the invention can be prokaryotic cells such as bacterial cells or eukaryotic cells such as plant cells or animal cells. The cells can be normal cells, immortalized cells, diseased 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 obtained from a natural source.

[0089] Particle size means the average diameter of the particles. If a particle size 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 scattering.

[0090] An inert atmosphere is generated by filling the respective containers or apparatuses 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, generating an inert atmosphere means, for example, by introducing liquid nitrogen or nitrogen, reducing the oxygen concentration to less than 10% (v / v) absolute.

[0091] Different types of mills are known to those skilled in the art.

[0092] A pin mill, also known as a centrifugal impact mill, crushes solids, whereby the pins protruding from a high-speed rotating disk provide the crushing energy. Pin mills are sold, for example, by Munson Machinery (USA), Premium Pulman (India) or Sturtevant (USA).

[0093] Jet mills use compressed gas to accelerate the particles, causing them to collide with each other in the process chamber. Jet mills are sold, for example, by Sturtevant (USA) or PMT (Austria).

[0094] The Fitz mill, commercialized by Fitzpatrick (USA), uses a rotor with blades for grinding.

[0095] A process that runs continuously is a process that does not run batchwise. If a grinding process runs continuously, it means that the culture medium components are continuously and steadily fed into the mill over a certain period of time.

[0096] The cell culture medium according to the invention, in particular a complete medium, generally 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.

[0097] The culture medium may also contain sodium pyruvate, insulin, plant protein, fatty acids and / or fatty acid derivatives and / or pluronic acid and / or surface-active components, such as chemically prepared non-ionic surfactants. An example of a suitable non-ionic surfactant is, for example, a bifunctional block copolymer surfactant with a primary hydroxyl end, available from BASF, Germany under the trade name pluronic which is also known as poloxamer.

[0098] 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).

[0099] Examples of the amino acids according to the invention are tyrosine, protein amino acids, especially the essential amino acids leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, and non-protein amino acids, preferably L-amino acids.

[0100] Tyrosine means L- or D-tyrosine, preferably L-tyrosine.

[0101] Cysteine means L- or D-cysteine, preferably L-cysteine.

[0102] Examples of vitamins are vitamin A (retinol, retinal, various retinols and four carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, nicotinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxol, pyridoxamine, pyridoxal), vitamin B7 (biotin), vitamin B9 (folic acid, folinic acid), vitamin B 12 (cyanocobalamin, hydroxocobalamin, methylcobalamin), vitamin C (ascorbic acid), vitamin D (ergocalciferol, cholecalciferol), vitamin E (tocopherols, tocotrienols) and vitamin K (phylloquinone, menaquinone). Also included are vitamin precursors.

[0103] 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 (CuSO4·5H2O), sodium chloride (NaCl), calcium chloride (CaCl2·2H2O), potassium chloride (KCl), iron(II) sulfate, anhydrous sodium dihydrogen phosphate (NaH2PO4), anhydrous magnesium sulfate (MgSO4), anhydrous disodium hydrogen phosphate (Na2HPO4), magnesium chloride hexahydrate (MgCl2·6H2O), zinc sulfate heptahydrate.

[0104] Examples of buffers are CO2 / HCO3 (carbonate), phosphate, HEPES, PIPES, ACES, BES, TES, MOPS and TRIS.

[0105] Examples of cofactors are thiamine derivatives, biotin, vitamin C, NAD / NADP, cobalamin, flavin mononucleotide and derivatives, glutathione, nucleotides, phosphates and derivatives.

[0106] According to the present invention, the 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.

[0107] Compared with the complete medium, the feeding medium may have a different composition. They usually contain amino acids, trace elements and vitamins. They may also contain a sugar component, but sometimes for production reasons, the sugar component is added in a separate feed.

[0108] A suitable feeding medium may contain, for example, one or more of the following compounds:

[0109] L-asparagine monohydrate

[0110] L-isoleucine

[0111] L-phenylalanine

[0112] L-sodium glutamate monohydrate

[0113] L-leucine

[0114] L-threonine

[0115] L-lysine monohydrochloride

[0116] L-proline

[0117] L-serine

[0118] L-arginine monohydrochloride

[0119] L-histidine monohydrochloride monohydrate

[0120] L-methionine

[0121] L-valine

[0122] L-sodium aspartate monohydrate

[0123] L-tryptophan

[0124] Choline chloride

[0125] Inositol

[0126] Nicotinamide

[0127] D(+)-calcium pantothenate

[0128] Pyridoxine hydrochloride

[0129] Thiamine hydrochloride

[0130] Micronized vitamin B12 (cyanocobalamin)

[0131] Biotin

[0132] Folic acid

[0133] Riboflavin

[0134] Magnesium sulfate anhydrous

[0135] Copper(II) sulfate pentahydrate

[0136] Zinc sulfate heptahydrate

[0137] 1,4-Diaminobutane dihydrochloride

[0138] Ammonium heptamolybdate tetrahydrate

[0139] Cadmium sulfate hydrate

[0140] Manganese(II) chloride tetrahydrate

[0141] Nickel(II) chloride hexahydrate

[0142] Sodium metasilicate

[0143] Sodium metavanadate

[0144] Tin(II) chloride dihydrate

[0145] Sodium selenite (approx. 45% Se)

[0146] Sodium dihydrogen phosphate monohydrate

[0147] Ammonium iron(III) citrate (approx. 18% Fe)

[0148] Freezing according to the invention means cooling to a temperature below 0 °C.

[0149] The gist of the present invention is to provide a powdered cell culture medium, such as a dry powder medium prepared by dry compaction, which can be easily dissolved in a suitable solvent by simply mixing the powder and the solvent, such that the powder dissolves and a liquid cell culture medium suitable for culturing cells is produced, such as a complete medium, a medium supplement, a subgroup of a medium or a feed, which has the required and homogeneous concentration of medium components.

[0150] The simple dissolution of the powdered cell culture medium 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 can be dissolved in 1 liter of water. However, the concentration of tyrosine required in the cell culture medium is often higher. Additionally, the solubility of leucine at approximately 22.1 g / kg at 25 °C and the solubility of isoleucine at 32.4 g / kg at 25 °C are often considered insufficient.

[0151] It has been found that, on the one hand, N-lactyl derivatives of amino acids generally have higher solubility in aqueous solutions and, on the other hand, can be used as substitutes for the respective amino acids and are equally suitable as components of cell culture media as the corresponding natural amino acids.

[0152] Preferably, in order to achieve optimal performance, the cell culture medium according to the invention comprises natural amino acids as well as N-lactyl amino acids. In the case where the medium is a feed medium or another medium additive added to a basal medium comprising natural amino acids, the feed medium or medium additive may comprise only N-lactyl amino acids and not the corresponding natural amino acids.

[0153] Typically in the basal medium and the perfusion medium, the molar ratio between the N-lactyl amino acid and the corresponding natural amino acid is from 5:1 to 1:5, preferably from 5:1 to 1:1.

[0154] The overall concentration of each lactyl amino acid in the ready-to-use liquid basal / perfusion medium as well as in the feed medium or medium additive is very flexible. The upper limit is only limited by the solubility of the lactyl amino acid in the respective medium. Thus, it is generally possible to produce liquid media with lactyl amino acid concentrations up to 500 mmol / l or more.

[0155] The solubility of the N-lactyl amino acids is higher than that of the respective natural amino acids (see Tables 1 and 2 in Example 2).

[0156] In order to even further increase the solubility of the N-lactyl amino acids, salts can be formed by reacting the derivatives with suitable bases. Sodium salts are preferred.

[0157] The cell culture medium according to the invention can comprise one or more, which means for example one, two, three, four, five, six, seven, eight, nine or ten N-lactyl amino acids.

[0158] The powdered cell culture medium of the invention is preferably produced by mixing all the components and grinding them. The mixing of the components is known to the person skilled in the art of producing dry powder cell culture media by grinding. Preferably, all the components are thoroughly mixed such that all parts of the mixture have substantially the same composition. The higher the degree of uniformity of the composition, the better the quality of the resulting medium with respect to homogeneous cell growth.

[0159] The grinding can be carried out with any type of mill suitable for producing powdered cell culture media. Typical examples are ball mills, pin mills, Fitz mills or jet mills. Preferred are pin mills, Fitz mills or jet mills, very preferably a pin mill.

[0160] The person skilled in the art knows how to operate such mills.

[0161] In the case of a large-scale equipment mill with a disk diameter of about 40 cm, for example, a needle mill usually operates at 1 - 6500 revolutions per minute, preferably 1 - 3000 revolutions per minute.

[0162] Grinding can be accomplished under standard grinding conditions, resulting in a powder with a particle size of 10 to 300 μm, most preferably 25 to 120 μm.

[0163] Preferably, all components of the mixture to be ground are dry. This means that if they contain water, they only contain water of crystallization, but no more than 10%, preferably no more than 5%, and most preferably no more than 2% by weight of unbound or uncoordinated water molecules.

[0164] In a preferred embodiment, the grinding is carried out in an inert atmosphere. The preferred inert protective gas is nitrogen.

[0165] In another preferred embodiment, all components of the mixture are frozen before grinding. Freezing of the components before grinding can be accomplished by any means that ensures the components are cooled to a temperature below 0 °C and most preferably below -20 °C. In a preferred embodiment, the freezing is done with liquid nitrogen. This means treating the components with liquid nitrogen, for example, by pouring liquid nitrogen into the container in which the components are stored before introducing them into the mill. In a preferred embodiment, the container is a feeder. If the container is a feeder, the liquid nitrogen is preferably introduced on or near the side of the feeder where the components are introduced.

[0166] Typically, the components are treated with liquid nitrogen for 2 to 20 seconds.

[0167] Preferably, the cooling of the components is done in such a way that all components entering the mill are at a temperature below 0 °C, most preferably below -20 °C.

[0168] In a preferred embodiment, all components are placed in a container, and the mixture is transferred from the container to a feeder, most preferably to a metering screw feeder. In the feeder, the components are sometimes further mixed - depending on the type of feeder - and additionally cooled. Then the cooled mixture is transferred from the feeder to the mill such that the mixture being ground in the mill preferably still has a temperature below 0 °C, more preferably below -20 °C.

[0169] Typically, the mixing time, which means the residence time of the component mixture in the feeder, is more than one minute, preferably 15 to 60 minutes.

[0170] The metering screw feeder, also known as the dosage snail, usually operates at a speed of 10 to 200 revolutions per minute, preferably it operates at 40 to 60 revolutions per minute.

[0171] Typically, the temperature of the mill is maintained between -50 and +30 °C. In a preferred embodiment, the temperature is maintained at about 10 °C.

[0172] The oxygen level during grinding is preferably below 10% (v / v).

[0173] The process can be run, for example, batchwise or continuously. In a preferred embodiment, the process according to the invention is carried out continuously by continuously filling a mixture of components into a feeder for cooling over a certain period of time and continuously filling the cooled mixture from the feeder into the mill.

[0174] After grinding, the resulting dry powder culture medium can be further compacted, for example, by dry compaction in a roll press to increase the particle size.

[0175] For use of the powder culture medium, a solvent, preferably water (most particularly distilled water and / or deionized water or purified water or water for injection) or an aqueous buffer is added to the culture medium, and the components are mixed until the culture medium is completely dissolved in the solvent, and a ready-to-use liquid culture medium is produced.

[0176] The solvent can also contain saline, soluble acid or base ions providing a suitable pH range (usually in the range between pH 1.0 and pH 10.0), stabilizers, surfactants, preservatives and alcohols or other polar organic solvents.

[0177] It is also possible to add further substances, such as buffer substances for adjusting the pH, fetal bovine serum, sugars, etc., to the mixture of cell culture medium and solvent. The resulting liquid cell culture medium is then brought into contact with the cells to be grown or maintained.

[0178] Although culture medium compositions containing a higher concentration of one or more natural amino acids will show turbidity when mixed with a solvent due to undissolved amino acids, the cell culture medium according to the invention in which the amino acids are completely or partially replaced by the corresponding N-lactyl amino acids gives a clear solution, as shown by the turbidity measurements in Example 3 and Figure 2 and Figure 3 as shown in the turbidity measurements in

[0179] The invention further relates to a process for culturing cells by:

[0180] a) providing a bioreactor

[0181] b) mixing the cells to be cultured with the cell culture medium according to the invention

[0182] c) incubating the mixture of step b).

[0183] In one embodiment, the bioreactor is a perfusion bioreactor.

[0184] A bioreactor is any vessel or tank in which cells can be cultured. Incubation is typically done under suitable conditions such as suitable temperature and the like. Those skilled in the art know the suitable incubation conditions for supporting or maintaining cell growth / culture.

[0185] A perfusion bioreactor is a bioreactor in which perfusion cell culture can be carried out. It includes a bioreactor vessel that is usually closed during cell culture, a stirrer in the vessel, a pipeline for introducing fresh culture medium, a harvest pipeline for withdrawing a harvest stream containing cells, liquid culture medium and target product from the bioreactor, and a cell retention device in the harvest pipeline that retains cells while allowing the liquid portion of the harvest to be collected. A review on perfusion cell culture providing details on favorable setups 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.

[0186] It has been found that the present invention is also well-suited for the preparation of feed media. Due to limitations in the availability of certain amino acids, especially in terms of the concentrations required for feed media, feed media cannot be prepared at the required high concentrations, or they need to be prepared under extreme pH conditions such as extremely alkaline pH. This can negatively affect the nutrient supply to cells and, to some extent, accelerate cell death by exposure to extremely alkaline pH values.

[0187] Thus, there is a need here for a feed medium that contains all the required components at a high concentration in a single feed. Additionally, the pH of the feed should not negatively affect cell culture.

[0188] It has been found that N-lactyl amino acids have improved solubility and can replace the corresponding natural amino acids in highly concentrated feed media without any negative effect on cell growth and / or productivity at a pH below 8.5, and sometimes even have a positive effect.

[0189] Accordingly, the present invention also relates to a feed medium in the form of a powdered medium or a liquid medium after dissolution.

[0190] The resulting liquid medium contains one or more N-lactyl amino acids, usually at a concentration higher than 10 mmol / l or even higher than 50 mmol / l, and preferably has a pH of 8.5 or lower.

[0191] In a preferred embodiment, the pH is from 6.5 to 7.8.

[0192] The present invention also relates to a process for culturing cells in a bioreactor by:

[0193] - filling the bioreactor with cells and an aqueous cell culture medium

[0194] - incubating the cells in the bioreactor

[0195] - adding the cell culture medium to the bioreactor continuously throughout the time or one or more times during the cell incubation time, the added cell culture medium preferably having a pH of less than 8.5 and containing at least one N-lactyl amino acid. Usually, the culture medium contains 50 to 150 g / l of solid components dissolved in a solvent.

[0196] In one embodiment, the added culture medium is a feed medium and the process is a fed-batch process. In another medium, the added culture medium is a perfusion medium and the process is a perfusion process.

[0197] It has been found that by using one or more N-lactyl amino acids, a feed medium containing all the necessary feed components at a high concentration (total concentration of 100 to 250 g / l) can be obtained. In contrast to known processes in which two or more different feed media need to be fed into the bioreactor, the present invention provides a culture medium and a method which enable the use of a single feed medium which contains all the components at a high concentration. Additionally, the pH of the feed medium according to the present invention is generally below 8.5.

[0198] In a preferred embodiment, in the process of the present invention, the feed medium added to the bioreactor continuously throughout the incubation or one or more times during said time always has the same composition.

[0199] The present invention is further illustrated by the following figures and examples, however, it is not limited thereto.

[0200] The entire disclosures of all applications, patents and publications cited above and below, as well as the corresponding European patent application EP 19209150.2 filed on November 14, 2019 are hereby incorporated by reference. Examples

[0201] The following examples represent the practical application of the present invention.

[0202] Example 1: Synthesis of L-lactyl-L-phenylalanine

[0203] The synthesis of L-lactyl-L-phenylalanine starts 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): pp. 582-587). First, L-phenylalanine methyl ester hydrochloride is synthesized: 2.00 g of L-phenylalanine (12.05 mmol) is dissolved in 100 mL of methanol and kept under continuous stirring in an ice bath; SOCl2 is slowly added until the final concentration is 1 M. The reaction is monitored by TLC (eluent: n-butanol: acetic acid: H2O, 4:1:1 by volume; UV and ninhydrin detection, Rf = 0.6) and occurs overnight. The reaction mixture is dried under vacuum, methanol is added and evaporated again under reduced pressure (4 times) to completely eliminate HCl; the crude yield is 98%. Then, (S)-2-acetoxypropanoic acid (0.41 g, 3.12 mmol) is dissolved together with 1.12 g (2.96 mmol) of HBTU in 4 mL of CH2Cl2, and the mixture is kept under continuous stirring at room temperature for 30 minutes to activate the carboxyl functional group. The previously synthesized L-phenylalanine methyl ester hydrochloride (0.68 g, 3.12 mmol) is dissolved together with DIPEA (1.54 mL, 9.36 mmol) in 4 mL of CH2Cl2, and then added to the activated acetoxypropanoic acid. The reaction is left to stand at room temperature with magnetic stirring for 4 hours. The reaction is monitored by TLC (eluent: ethyl acetate; UV absorbance detector, R f = 0.8). Then, the organic solution is washed with saturated solutions of KHSO4 (3 times) and NaHCO3 (3 times), dried over MgSO4 and filtered, and the product is dried under vacuum; the crude yield is 46%. Then, the methyl and acetyl protecting groups are removed by reacting the product at 0 °C in a mixture of 0.34 g (1.296 mmol) of Ba(OH)2·5H2O in 20 mL of tetrahydrofuran (THF) / H2O (1:1, v / v) for 20 minutes. Then, THF is eliminated under vacuum, and the aqueous solution is acidified to pH 3.0 with HCl. The resulting solution is analyzed by LC / ESI-MS.

[0204] Example 2: Lac-Ile and Lac-Leu have increased solubility in water compared to their respective amino acids By preparing saturated solutions, the maximum solubility of Ile and Leu is compared with the solubility of their respective lactyl derivatives or their salts in water at 25 °C. After sedimentation, the solution is dried using infrared rays (120 °C, 120 minutes), and the residual mass is determined in g / kg.

[0205] As shown in Table 1, the solubility of lactyl-AA and its salts is significantly higher when compared to the solubility of their respective amino acids in water. For Leu, the increase in solubility is approximately 30-fold for Lac-Leu (N-lactyl leucine), while when compared to Ile, the increase in solubility is 20-fold for Lac-Ile (N-lactyl isoleucine). To rule out that the increase in solubility is due to the sodium salt form of lactyl-AA, separate experiments were performed to compare the solubility 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, indicating that as expected, the formation of the sodium salt has increased the solubility of Leu, but the increase in solubility obtained with the lactyl derivative is significantly more important and thus cannot be due solely to the salt form. The same behavior is thought to be valid for other lac-AA. In summary, these results indicate that lac-AA and its salts are suitable candidates to increase the solubility of cell culture media and feed formulations by replacing their respective amino acids.

[0206] Table 1: Solubility of amino acids and their respective lac-AA or their salts in water at 25 °C. Solubility experiments were performed using saturated solutions and determination of the residual mass after infrared drying.

[0207]

[0208] Example 3: In an Ile- and Leu-depleted 4Feed, the maximum solubility of Lac-AA when compared to their respective amino acids

[0209] Increasing amounts of Lac-Leu and Lac-Ile and their salts were added to an Ile- and Leu-depleted cell culture feed formulation ( 4Feed). Similarly, increasing amounts of Ile and Leu were added to the same feed formulation as a control. 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 an amino acid or Lac-AA, the feed was agitated for 10 minutes and the turbidity was measured. The experiments were performed at room temperature (25 °C).

[0210] It was found that the maximum solubility of Ile in the Ile / Leu-depleted 4Feed was approximately 105 mM, while for lac-Ile, the maximum tested concentration of 951 mM was still soluble, with a turbidity value below 5 NTU ( Figure 2 ). This indicates that the solubility of Lac-Ile in the Ile / Leu-depleted 4Feed is at least 9 times that of Ile.

[0211] Figure 2 It is shown that in the case of depletion of Ile and Leu Determination of the maximum solubility of Ile or Lac-Ile in 4Feed formulation (113 g / L, pH 7.0 + / - 0.2). Solutions with a turbidity below 5 NTU were considered soluble.

[0212] It was found that in the depletion of Ile and Leu The maximum solubility of Leu in 4Feed is about 90 mM, while for Lac-Leu, the maximum soluble concentration (with a turbidity value below 5 NTU) is about 600 mM ( Figure 3 ). This indicates that Lac-Leu is depleted of Ile / Leu The solubility in 4Feed is 6.6 times that in Leu.

[0213] Figure 3 It is shown that in the case of depletion of Ile and Leu Determination of the maximum solubility of Leu or Lac-Leu in 4Feed formulation (113 g / L, pH 7.0 + / - 0.2). Solutions with a turbidity below 5 NTU were considered soluble.

[0214] Example 4: Use of Lac-AA enables concentration of cell culture medium formulations at neutral pH.

[0215] The maximum solubility of 4Feed (normal concentration 130 g / L) was determined by dissolving increasing amounts of feed dry powder medium in water until precipitation was visually detected. For each condition, the feed was stirred for about 30 minutes, the pH was adjusted to 7.0 + / - 0.2, and the solution was stirred for an additional 10 minutes for equilibrium. Osmotic pressure and turbidity were measured, and photos were taken ( Figure 4 ). The data indicated that the 1.2x concentrate (160 g / L) of this formulation was no longer soluble, as particles could be detected in the suspension and the turbidity was well above the limit of 5 NTU.

[0216] Figure 4 Shows Solubility limit of 4Feed at pH 7.0. Turbidity was measured using a turbidimeter.

[0217] Since Ile and Leu have been identified 4Feed preparations were concentrated in the first limiting amino acid, thus producing a new backbone feed depleted of Ile and Leu ( 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 + lactyl derivatives 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.

[0218] The results indicated that the maximum solubility of the Ile / Leu-depleted 4Feed supplemented with Lac-Leu and Lac-Ile was obtained between 189 g / L and 212 g / L. 4Feed ( Figure 5 ). Considering that 4Feed (containing Ile and Leu) had a concentration of 130 g / L, which represents an approximately 50% increase in concentration when Ile and Leu are replaced by Lac-Ile and Lac-Leu.

[0219] Figure 5 The turbidity of solutions containing increasing amounts of Ile / Leu-depleted 4Feed supplemented with Lac-Leu and Lac-Ile derivatives (equimolar concentration compared to free AA) is shown.

[0220] Example 5: Lac-Leu and Lac-Ile are stable when stored in the dark at 4 °C or RT for 3 months in Cellvento 4Feed-Ile / Leu.

[0221] 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 in 4Feed–Ile / Leu pH 7.0 were performed from 100 mM to 100 μM to determine the linearity of the method. A 200-fold dilution was performed in water prior to LC-MS analysis. The method was developed on a UHPLC (Vanquish, Thermo Fisher) coupled to an ESI-Q-ToF mass spectrometer (Impact II, Bruker Daltonics).

[0222] Briefly, 1 μL of the sample was loaded onto an XSelect HSS T3 column (2.1 x 150 mm, 3.5 μm, Waters) thermostated at 40 °C with a flow rate of 300 μL / minute 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.

[0223] Table 2: Gradient of the chromatography method using XSelect HSS T3 column

[0224]

[0225] 1 : 20 mM ammonium formate / 0.1% FA; 2 : 100% methanol

[0226] LC-MS analysis was performed using an Impact II mass spectrometer equipped with an ESI source (Bruker Daltonics). MS acquisition was carried out in negative mode with end plate offset and capillary voltage set at 500 and 3500 V, respectively. The nebulizer and drying gas (250 °C) were set at 1.4 bar and 9.0 L / min, respectively. MS spectra were acquired in the m / z range 20 - 1000 at a scan rate of 5 Hz. Calibration was performed using a sodium formate solution injected at the start of the analysis.

[0227] The standard curves obtained for Lac-Leu and Lac Ile are presented in Figure 6 . Very good linearity was obtained for injections between 10 pmol and 400 pmol on column (500 μM to 20 mM in the feed), while no linearity was obtained for the range 2 pmol to 2 nmol on column (100 μM to 100 mM in the feed, not shown). Therefore, Lac-AA quantification should be performed by injecting 10 to 400 pmol on column.

[0228] Figure 6 Shows the linearity obtained by the LC-MS method for Lac-Leu and Lac-Ile in 4Feed–Ile / Leu after 200x dilution in water. Very good linearity was obtained in the range 10 - 400 pmol on column.

[0229] Using the previously developed method, the stability of Lac-Ile and Lac-Leu in 4Feed-Ile / Leu was monitored in samples stored in the dark at 4 °C and RT for 3 months. As shown in the figure, the stability of the two lactyl AAs at 4 °C and RT is very good, indicating that Lac-Ile and Lac-Leu are stable even when the feed formulation is stored in the dark at 4 °C or RT for up to 3 months.

[0230] Figure 7 Shows the stability of Lac-Ile and Lac-Leu in 4Feed-Ile / Leu as determined by LC-MS.

[0231] Example 6: Lac-Ile and Lac-Leu can replace their respective amino acids in the feed. Cell culture results using a CHOK1GS clone producing IgG1.

[0232] For the cell culture experiment, a CHOK1GS suspension cell line expressing human IgG1 was used. The cells were cultured in quadruplicate in 4CHO medium (Merck Darmstadt, Germany) using 50 mL spinner flasks, with an initial culture volume of 30 mL and an inoculation density of 2x10 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 (Ile- and leu-depleted 4Feed) to replace 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 a feed depleted of the respective amino acids and without the addition of Lac-AA. Feeds were added at the following v / v ratios (3, 3, 6, 3, 3, and 3%) on days 3, 5, 7, 10, 12, and 14. 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.

[0233] 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 acids were quantified via UPLC after derivatization using a kit. Derivatization, chromatography, and data analysis were performed following the supplier's recommendations (Waters, Milford, MA).

[0234] When considering viable cell density ( Figure 8 ), the negative control with a feed depleted of Leu and Ile showed a rapid decrease in VCD after day 7, indicating that the cells require both amino acids for efficient growth. Compared to the positive control, replacing Ile with Lac-Ile had no effect on the peak VCD or the overall VCD profile, while replacing Leu with Lac-Leu had a positive effect on the duration of the plateau phase, as the cells remained at approximately 15,000,000 cells / mL for several days from day 10 to day 14.

[0235] Figure 8 VCD within a 17-day fed-batch process is shown, where Lac-Leu or Lac-Ile replaced Leu and Ile in the feed, respectively. Depleted 4Feed is a negative control and contains no Leu or Ile.

[0236] For all conditions ( Figure 9 ), the IgG concentration obtained on day 14 was around 3 g / L, and for the conditions where Leu and Ile were replaced with Lac-Leu and Lac-Ile respectively, had slightly higher but not significantly higher titers.

[0237] Figure 9 Shows IgG produced within a 17-day fed-batch process, where Lac-Leu or Lac-Ile replaced Leu and Ile in the feed respectively.

[0238] Since lactate is released from the formation of free AA from Lac-AA, this metabolite in the supernatant was monitored during the duration of the FB process ( Figure 10 ). Higher lactate concentrations were observed after day 5 under the conditions where Leu and Ile were replaced with Lac-Leu and Lac-Ile respectively, most likely due to cleavage of the derivative. When calculating the area under the curve of the lactate concentration, the overall increase in lactate was quantified. Replacing Leu with Lac-Leu resulted in a 20% increase in free lactate, while replacing Ile with Lac-Ile resulted in a 37% increase, which was very similar to the lactate increase (36.5%) observed in the negative control.

[0239] Figure 10 Shows lactate production during a 17-day fed-batch process, where Lac-Leu or Lac-Ile replaced Leu and Ile in the feed respectively.

[0240] The concentration of amino acids was determined in the spent medium. Under the condition where Leu was replaced with Lac-Leu, the Leu concentration in the spent medium ( Figure 11 ) decreased very rapidly until day 7 and then increased again, suggesting that cleavage of Lac-AA takes time, most likely depending on the release or activation of specific enzymes (a slow-release technique similar to the previously developed phosphotyrosine release technique). Under the condition where Ile was replaced with Lac-Ile, the overall leucine concentration in the fed-batch decreased moderately, indicating that other branched-chain amino acids can be used instead of Ile before effective cleavage of the lactyl derivative.

[0241] When considering Ile in the spent medium ( Figure 12) Similar behavior to 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, most likely depending on the release or activation of specific enzymes. Under the condition where Leu was replaced with Lac-Leu, the Ile concentration decreased after day 7, which also indicated that other branched-chain amino acids could be used instead of Leu.

[0242] Figure 11 : Quantification of Leu in the spent medium during a 17-day fed-batch process with Leu and Ile in the feed replaced with Lac-Leu and Lac-Ile, respectively.

[0243] Figure 12 : Quantification of Ile in the spent medium during a 17-day fed-batch process with Leu and Ile in the feed replaced with Lac-Leu and Lac-Ile, respectively.

[0244] The antibody quality produced in a control fed-batch process (using a feed containing Ile and Leu) was compared with that produced using a feed depleted of Leu or Ile and supplemented with Lac-Leu or Lac-Ile.

[0245] Using Protein A (PhyNexus Inc, San Jose, CA), antibodies were purified from cell culture supernatants. According to the manufacturer's instructions, after derivatization using the Rapid N-Glycan Sample Preparation kit (Prozyme, Hayward, CA) with 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS), the glycosylation pattern was analyzed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF). Briefly, the purified antibodies were denatured and immobilized, and the glycans were released by treatment with Digestion was released from the antibody and subsequently labeled with APTS at 50 °C for 60 minutes. After a cleaning step to remove the remaining APTS, the relative amounts of glycans were determined using a Pharmaceutical Analysis System CESI8000 Plus (Sciex, Washington, USA) with a LIF detector (Ex: 488 nm, Em: 520 nm). Separation was performed in a polyvinyl alcohol-coated capillary (total length: 50.2 cm, inner diameter: 50 μm) and 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 inlet and outlet buffer vials were changed every 20 cycles. The sample was introduced by injection at a pressure of 0.5 psi for a total of 12 seconds, followed by a soak step of 0.2 minutes to clean the capillary tip. Finally, separation was performed at 20 kV for 20 minutes, with a 0.17-minute ramp with reverse polarity applied. Peaks were identified based on their individual migration times and integrated according to the following parameters: peak width 0.05, threshold 10,000, and shoulder sensitivity 9,999.

[0246] 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.

[0247] Charge variants were measured using cIEF on a Capillary Electrophoresis CESI 8000 (Beckman Coulter / Sciex) 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 different pH markers, cathode / anode stabilizers, 3 M UreacIEF gel, and Pharmalyte.

[0248] For glycosylation ( Figure 13 ), high molecular weight species and low molecular weight species ( Figure 14 ), and charge variants ( Figure 15)The results obtained indicated no difference between the control condition and the condition in which Ile and Leu were exchanged with Lac-Ile and Lac-Leu, indicating that the amino acid exchange had no effect on the three key quality attributes of the IgG1 produced in this study.

[0249] Figure 13 Shows the glycosylation of the IgG1 produced in the control process or in the process in which the feed supplemented with Lac-Leu or Lac-Ile after depleting Ile / Leu was used. The glycan profile was determined using APTS labeling and CGE-LIF detection.

[0250] Figure 14 Shows the aggregation and fragmentation of the IgG1 produced in the control process or in the process in which the feed supplemented with Lac-Leu or Lac-Ile after depleting Ile / Leu was used. High molecular weight substances (HMW) and low molecular weight substances (LMW) were determined using size exclusion chromatography.

[0251] Figure 15 Shows the charge variants of the IgG1 produced in the control process or in the process in which the feed supplemented with Lac-Leu or Lac-Ile after depleting Ile / Leu was used. The charge variant distribution was determined using cIEF on Capillary Electrophoresis CESI8000.

[0252] Example 7: Confirm the performance of Lac-Leu and Lac-Ile using the CHOD G44 clone producing IgG1.

[0253] By performing fed-batch experiments with the CHOD G44 clone, the applicability of the present invention to different bioprocesses was confirmed. The results ( Figure 16 、 17 、18) indicated similar VCD and IgG titers for the Lac-Leu and Lac-Ile conditions compared to the control positive control. In contrast, the depleted 4Feed condition led to an early decrease in VCD and a significantly reduced titer after day 7. Data on the spent medium indicated that for this cell line, no increase in lactate cleavage from the Lac derivatives was detected. The explanation for this behavioral difference between the CHO-K1 GS cells and this DG44 cell line is currently unclear. The concentrations of Leu and Ile in the spent medium ( Figure 19 and) decreased during the first day of the FB culture and finally increased again after D7. This behavior was similar to that seen for the CHO-K1 GS cells and indicated a slow release process of the free AAs from the derivatives.

[0254] Figure 16 , 17 and 18 show the performance of the Lac-Leu and Lac-Ile processes compared to a control of the CHODG44 cell line expressing IgG1. Viable cell density ( Figure 16 ), IgG titer ( Figure 17 ), lactate concentration in the spent medium ( Figure 18 ).

[0255] Figure 19 and 20 show the relative concentrations of isoleucine ( Figure 19 ) and leucine ( Figure 20 ) in the spent medium in the Lac-Leu and Lac-Ile processes (CHODG44 cells) compared to a normal process using unmodified Ile and Leu.

Claims

1. A dry powder cell culture medium, which comprises at least one N-lactyl amino acid and / or its salt, wherein the N-lactyl amino acid is N-lactyl leucine and / or N-lactyl isoleucine and / or its salt.

2. The cell culture medium according to claim 1, characterized in that, The cell culture medium comprises one or more components of formula I: wherein R1 + is H + or Na + 、K + 、Mg 2+ 、Ca 2+ 、Li + , R2 is a characteristic residue of an amino acid.

3. The cell culture medium according to claim 1 or 2, characterized in that The cell culture medium comprises one or more sodium salts of N-lactyl amino acids.

4. The cell culture medium according to claim 1 or 2, characterized in that The cell culture medium comprises one or more N-lactyl amino acids and the corresponding amino acids and / or their salts.

5. The cell culture medium according to claim 1 or 2, characterized in that The culture medium comprises one or more N-lactyl amino acids and / or their salts, but does not comprise the corresponding amino acids and / or their salts.

6. A method for producing a cell culture medium according to any one of claims 1 to 5 by the following steps: 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 grinding.

7. A method for culturing cells by the following steps: a) Providing a bioreactor b) Mixing the cells to be cultured with a cell culture medium comprising at least one N-lactyl amino acid and / or its salt c) Incubating the mixture of step b), wherein the N-lactyl amino acid is N-lactyl leucine and / or N-lactyl isoleucine and / or its salt, and the cells are CHO cells.

8. The method according to claim 7, wherein The bioreactor is a perfusion bioreactor.

9. A method for culturing cells in a bioreactor by the following steps: - 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 throughout the time or one or more times during the cell incubation time whereby the culture medium comprises at least one N-lactyl amino acid and / or its salt, wherein the N-lactyl amino acid is N-lactyl leucine and / or N-lactyl isoleucine and / or its salt, and the cells are CHO cells.

10. The method according to claim 9, characterized in that The method is a fed-batch method, and the added culture medium is a feed medium having a pH lower than 8.5 and comprising at least one N-lactyl amino acid and / or its salt at a concentration higher than 10 mmol / l.

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