Cell culture methods

Abstract

To provide a cell culture method, production of proteins such as monoclonal antibodies in mammalian cells, a novel biomarker, and a method for controlling protein production using the novel biomarker.SOLUTION: A method of cell culture includes (i) culturing mammalian cells in a cell culture medium, and (ii) maintaining at least one metabolite selected from aconitic acid (AA), leucinic acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI), and N-acetylputrescinium (NAP) below an inhibitory concentration in the cell culture medium for the at least one metabolite.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present disclosure relates to a cell culture method, production of proteins such as monoclonal antibodies in mammalian cells, novel biomarkers, and a method for controlling protein production using the novel biomarkers.

Background Art

[0002] Mammalian cell lines such as Chinese hamster ovary (CHO) cell lines are used as hosts for producing therapeutic antibodies in the biopharmaceutical industry. In the process of antibody production, for example, CHO cells tend to use much of the available nutrients for generating waste metabolites rather than the amounts required for their growth, showing inefficient and poorly controlled metabolism. In the later stages of the bioprocess, although the nutrient levels are sufficient to support growth and protein production, the cells stop growing and stop antibody production. Metabolites accumulated during the process are one of the main rate-limiting factors in cell proliferation and antibody production. In order to control the metabolism of CHO cells and improve productivity, it is extremely important to identify inhibitory waste metabolites and examine their pathways.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Identification of inhibitory metabolites and development of control strategies are required.

Means for Solving the Problems

[0004] In one aspect, a cell culture method includes: (i) culturing cells in a cell culture medium; and (ii) maintaining at least one metabolite selected from aconitic acid (AA), leucinic acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI), and N-acetylputrescinium (NAP) below the inhibitory concentration in the cell culture medium for the at least one metabolite.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0006] The above and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims. Chinese hamster ovary (CHO) cells remain the major workhorse cells for monoclonal antibody (mAb) production. To achieve high antibody productivity, the manufacturing process is carried out under high cell density conditions (> 10 million cells / mL). However, while the cell density reaches record highs, the productivity will stagnate because the concentration of secreted inhibitory metabolites is high. The accumulation of these inhibitory metabolites impairs cell growth, and it has been shown that the generation of waste by-products (such as ammonia) changes important product characteristics of recombinant proteins, thus posing a risk to the overall product quality. In this study, in addition to common inhibitors, novel metabolic biomarkers were identified and characterized by metabolomics using LC-MS. The workflow of waste inhibitor metabolomics was the identification of candidate substances, verification and quantification of inhibitory effects, and development of control strategies.

[0007] New biomarkers for protein production in CHO cells and other mammalian cells are aconitic acid (AA), more specifically aconitic acid (AA), homoisocitric acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI), and N-acetylputrescine (NAP). Guanosine monophosphate (GMP) and indole-3-carboxylic acid (ICA), which have been previously identified, are also identified in the studies described herein and can be combined with the newly described biomarkers herein.

[0008] This specification describes a cell culture method in which the concentration of at least one metabolite selected from aconitic acid (AA), homoisocitric acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI), and N-acetylputrescine (NAP) is maintained at a low level in the cell culture medium. In cell culture, particularly in high-density cell culture such as fed-batch and / or perfusion bioprocesses aimed at producing large amounts of a recombinant protein of interest, cell growth may be inhibited by the accumulation of metabolites such as AA, HICA, CMP, MSA, TRI, and / or NAP. The inhibitory effects of these metabolites can be limited by maintaining their concentrations in the cell culture medium below the level that inhibits cell growth, recombinant protein productivity, and product quality. Product quality includes, but is not limited to, glycosylation patterns, charge variants, aggregation, and fragmentation, all of which can be controlled by the methods described herein.

[0009] In one aspect, the cell culture method includes (i) culturing cells in a cell culture medium to initiate a cell culture process and (ii) maintaining at least one metabolite selected from AA, HICA, CMP, MSA, TRI, and NAP below the inhibitory concentration in the same cell culture medium for the at least one metabolite. In one aspect, the cells are mammalian cells.

[0010] Exemplary CHO cell lines include CHO K1, CHO GS, and DG44. Other mammalian cell lines include cell lines used to produce human therapeutic products such as HEK293 cells, HT-1080 cells, engineered T cells, and engineered natural killer cells.

[0011] The inhibitory amount of the metabolite can range from 1 nM to 50 mM, more specifically from 100 nM to 10 mM. When the metabolite is AA, the inhibitory concentration can be less than 100 μM, 440 μM, 880 μM, 3 mM, 5 mM, or 10 mM.

[0012] When the metabolite is HICA, the inhibitory concentration can be less than 10 μM, 23.5 μM, 47 μM, 100 μM, 1 mM, or 3 mM. When the metabolite is CMP, the inhibitory concentration can be less than 5 μM, 10 μM, 20 μM, 100 μM, 500 μM, or 1 mM.

[0013] When the metabolite is MSA, the inhibitory concentration can be less than 1 μM, 3.75 μM, 7.5 μM, 100 μM, 1 mM, or 3 mM. When the metabolite is TRI, the inhibitory concentration can be less than 0.1 μM, 0.35 μM, 0.7 μM, 100 μM, 1 mM, or 3 mM.

[0014] When the metabolite is NAP, the inhibitory concentration can be less than 0.1 μM, 0.3 μM, 0.6 μM, 100 μM, 1 mM, or 3 mM. In one aspect, the method includes measuring the concentration of at least one metabolite. The concentration of the metabolite can be measured by any method known to an expert in the analysis.

[0015] The concentration of the metabolite can be measured one or more times during cell culture. In one aspect, the concentration of the metabolite is measured continuously, intermittently, every 30 minutes, hourly, every 2 hours, twice a day, daily, or every two days. In a preferred aspect, the concentration of the metabolite is measured daily.

[0016] It is also possible to measure the concentration in a non-automated manner that is not integrated into the cell culture method. For example, a sample can be manually collected from the cell culture medium, and a measurement method for measuring a specific concentration in the sample can be used. Alternatively, determining the concentration of metabolites may be automated and integrated into the cell culture method.

[0017] Exemplary methods for measuring metabolite concentrations include nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, high-performance / ultra-high-performance liquid chromatography (H / UPLC), liquid chromatography-mass spectrometer (LC-MS), gas chromatography-mass spectrometer (GC-MS) techniques, or combinations thereof. For example, in GC-MS, experiments can be performed using an autosampler that collects samples from the reactor and transfers them to the instrument in a programmed manner. In data not shown, the identification of AA, HICA, CMP, MSA, TRI, and NAP was confirmed using one or a combination of the aforementioned methods.

[0018] In one aspect, when the measured concentration of one or more metabolites exceeds a predetermined value such as an inhibitory concentration, a control strategy for limiting the concentration of the inhibitory metabolite is used. In Control Strategy 1, the concentration of the precursor of at least one metabolite (e.g., amino acid, glucose) in the cell culture medium is reduced by reducing the amount of precursor provided to the cells. Limiting the precursor of the metabolite will mitigate the accumulation of the inhibitory metabolite. This method is also called media control. The predetermined value is selected such that the reduction in the concentration of the precursor prevents the concentration of one or more metabolites from rising above the inhibitory concentration.

[0019] When the metabolite is AA, the precursors are glutamine, glucose, arginine, and / or asparagine. When the metabolite is HICA, the precursors are leucine and / or isoleucine.

[0020] When the metabolite is CMP, the precursors are glutamine, arginine, and / or aspartate. When the metabolite is MSA, the precursors are lysine, isoleucine, serine, glucose, and / or glutamine.

[0021] When the metabolite is TRI, the precursors are aspartic acid, tryptophan, and / or glutamine. When the metabolite is NAP, the precursors are arginine, proline, aspartic acid, glutamine, and / or asparagine.

[0022] The aforementioned combinations of precursors can also be reduced in the method. Lowering the concentration of the precursor of the inhibitory metabolite can lower the concentration of the inhibitor for optimizing the process.

[0023] The concentration of the precursor in the cell culture medium can be reduced by decreasing the amount of precursor provided to the cells, for example, by decreasing the concentration of the precursor in the feed medium, by decreasing the feed rate, by decreasing the number or amount of feeds, or by a combination thereof. For example, the feed medium can be replaced with a feed medium containing a lower concentration of the precursor.

[0024] The concentration of the precursor can be in the range of 0.01 g / L to 2 g / L. More specifically: When the precursor is arginine, the concentration can be less than 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, or 0.6 g / L.

[0025] When the precursor is asparagine, the concentration can be less than 0.1 g / L, 0.3 g / L, 0.5 g / L, 0.7 g / L, or 0.9 g / L. When the precursor is aspartic acid, the concentration can be less than 0.1 g / L, 0.3 g / L, 0.5 g / L, 0.7 g / L, or 0.9 g / L.

[0026] When the precursor is glutamine, the concentration can be less than 0.1 g / L, 0.6 g / L, 0.9 g / L, 1.2 g / L, or 1.5 g / L. When the precursor is isoleucine, the concentration can be less than 0.05 g / L, 0.15 g / L, 0.25 g / L, 0.35 g / L, or 0.45 g / L.

[0027] When the precursor is leucine, the concentration can be less than 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, or 0.5 g / L. When the precursor is lysine, the concentration can be less than 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, or 0.5 g / L.

[0028] When the precursor is proline, the concentration can be less than 0.05 g / L, 0.15 g / L, 0.25 g / L, 0.35 g / L, or 0.45 g / L. When the precursor is serine, the concentration can be less than 0.05 g / L, 0.15 g / L, 0.25 g / L, 0.35 g / L, or 0.45 g / L.

[0029] When the precursor is tryptophan, the concentration can be 0.01 g / L, 0.03 g / L, 0.07 g / L, 0.1 g / L, or 0.15 g / L. Experimental designs such as Plackett-Burman Design or Response Surface Methodology (RSM) can be applied to find the optimal precursor concentration from the above concentration ranges to minimize the accumulation of inhibitory metabolites.

[0030] Reducing the amount of glucose can include culturing cells at a low glucose concentration, for example, by using alternative carbon sources including but not limited to fructose and galactose, using cell lines with reduced protein levels of glycolytic enzymes including but not limited to hexose transporters or lactate dehydrogenase, using cell lines with suppressed cellular protein levels of both lactate dehydrogenase and pyruvate dehydrogenase kinase, using cell lines with overexpression of pyruvate carboxylase enzyme, or using inhibitors (small molecule or protein-based) against signaling pathways (such as AKT, mTOR, HIF1a) that regulate the activity of energy metabolism pathways (glycolysis, TCA cycle, redox pathway).

[0031] In control strategies 2a and b and 3, upstream and downstream enzyme expression (strategy 2) or activity (strategy 3) is manipulated to alleviate the synthesis of inhibitory metabolites. The general idea behind control strategies 2 and 3 is to down-regulate upstream enzymes so that the cells produce less inhibitory metabolites and / or to up-regulate downstream enzymes so that the cells consume metabolites to generate energy. Figure 10 shows the three control strategies illustrated for NAP.

[0032] In control strategy 2a, the upstream and / or downstream enzyme expression that controls the metabolite level can be regulated using inhibitory nucleic acids such as siRNA. The term "inhibitory nucleic acid" refers to single-stranded or double-stranded RNA or DNA that can reduce or inhibit the expression of a target gene or sequence, specifically, triple-stranded oligonucleotides, ribozymes, aptamers, small interfering RNAs including siRNA (short interfering RNA) and shRNA (short hairpin RNA), antisense RNA or a part thereof, or RNAs such as their analogs or mimics. The inhibitory nucleic acid can act, for example, by mediating degradation or by inhibiting the translation of mRNA complementary to the interfering RNA sequence. When an inhibitory nucleic acid is administered to mammalian cells, the expression (e.g., transcription or translation) of the target sequence is reduced (e.g., by 5%, 10%, 25%, 50%, 75%, or 90 - 100%). Typically, a nucleic acid inhibitor comprises or corresponds to at least a part of the target nucleic acid molecule or its ortholog, or comprises at least a part of the complementary strand of the target nucleic acid molecule. The inhibitory nucleic acid can have substantial or complete identity to the target gene or sequence, or can contain mismatch regions (i.e., mismatch motifs). The sequence of the inhibitory nucleic acid can correspond to the full-length target gene or its sub-sequence. In one aspect, the inhibitory nucleic acid molecule is chemically synthesized.

[0033] In control strategy 2b, recombinant DNA is used to overexpress one or more downstream enzymes that control the metabolite level, thereby reducing the metabolite level. In control strategy 3, enzyme activation can be controlled by two different approaches.

[0034] Approach 1: By adding an inhibitor of enzyme activity, enzyme expression is regulated to reduce the synthesis of metabolites. Exemplary inhibitors of enzyme activity include small molecules, peptides, proteins, and nucleic acids that bind to the active site of the enzyme and interfere with its activity.

[0035] Approach 2: Since all inhibitory metabolites can be further metabolized into the TCA cycle to supply energy, glycolytic activators such as HK-1 and GK can be used to mitigate the accumulation of inhibitory metabolites.

[0036] For control strategies 2 and 3 in certain embodiments: The metabolite is AA, and the enzyme is ADI1, HOGA1, TAD1, or a combination thereof; The metabolite is HICA, and the enzyme is GOT1, D-HicDH, MMUT, AUH, HMGCL, HADHA / B, or a combination thereof; The metabolite is CMP, and the enzyme is UCK1 / 2, NT5, CMAS, CMPK1, DDYD, CDA, SLC35A1, RRM1, HOGA1, or a combination thereof; The metabolite is MSA, and the enzyme is GOT1, ETHE1, AMT, HADHA / B, MMUT, or a combination thereof; The metabolite is TRI, and the enzyme is NADSYN1, NNMT, CAT, NMNAT1, SULT4A1, or a combination thereof; The metabolite is NAP, and the enzyme is SAT1 / 2, HOGA1, AMD1, ODC1, GOT1, MAOB, or a combination thereof; or, the above combinations, is.

[0037] In control strategy 4, for at least one metabolite, maintaining the at least one metabolite in the cell culture medium below the inhibitory concentration involves controlling process parameters including temperature, dissolved oxygen level, pH, or a combination thereof. The optimal temperature, dissolved oxygen level, and pH can be determined by the rates of enzyme reaction and inhibitory metabolite accumulation. This process can be optimized for fed-batch and perfusion bioprocesses.

[0038] Exemplary temperatures are from about 30 °C to about 40 °C, specifically, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, and 37 °C in the early and late phases of cell culture.

[0039] Exemplary pH values are in the range of pH 5.5 to 8.5, for example, pH 6.5, 6.7, 6.9, 7, 7.2, 7.4, or 7.6. CO 2 spraying and / or Na 2 CO 3 The pH balance can be achieved using replenishment. Exemplary CO 2 spraying rates include 0.3 SLPH, 0.4 SLPH, 0.5 SLPH, 0.6 SLPH, and 0.7 SLPH (standard liter per hour). Na 2 CO 3 Exemplary stirring rates for replenishment include 90 RPM, 120 RPM, 150 RPM, and 170 RPM (revolutions per minute).

[0040] Exemplary dissolved oxygen set points are from 5% to 50%, for example, including 20%, 30%, and 40%. Any cell that can be grown in cell culture can be used in the methods described herein. In some embodiments, the cell is a mammalian cell. Non-limiting examples of mammalian cells include: BALB / c mouse myeloma strain (NSO / I, ECACC number: 85110503); human retinoblastoma cells (PER.C6, CruCell, Leiden, Netherlands); simian kidney CV1 strain transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney strain (293 or 293 cells subcloned for suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells + / − DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HeLa, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC5 cells; FS4 cells; and human hepatocellular carcinoma cell line (HepG2). In one embodiment, the cell is a CHO cell, a HEK293 cell, an HT-1080 cell, a genetically engineered T cell, and a genetically engineered natural killer cell. Genetically engineered T cells include CAR T cells.

[0041] Any number of commercially and non-commercially available hybridoma cell lines can be utilized by the methods described herein. As used herein, the term "hybridoma" refers to a cell or the progeny of a cell resulting from the fusion of an immortalized cell and an antibody-producing cell. The resulting hybridoma is an immortalized cell that produces an antibody. The individual cells used to generate the hybridoma can be derived from any mammalian source, including but not limited to rats, pigs, rabbits, sheep, goats, and humans. In some embodiments, the hybridoma is a trioma cell line that results when the progeny of a heterohybrid myeloma fusion, which is a product of the fusion of a human cell and a mouse myeloma cell line, is subsequently fused with a plasma cell. In some embodiments, the hybridoma is any immortalized hybrid cell line that produces an antibody, such as, for example, quadromas. One of ordinary skill in the art will understand that hybridoma cell lines can have different nutritional requirements and / or may require different culture conditions for optimal growth, and that the conditions can be modified as needed.

[0042] As used herein, the terms "culture" and "cell culture" refer to a population of cells suspended in a medium under conditions suitable for the survival and / or growth of the cell population. As will be apparent to one of ordinary skill in the art, in some embodiments, these terms as used herein refer to a combination that includes the cell population and the medium in which the population is suspended. In some embodiments, the cells of the cell culture include mammalian cells.

[0043] The methods described herein can be used with any cell culture method suitable for a desired process (e.g., the production of a recombinant protein (e.g., an antibody)). As a non-limiting example, the cells may be grown in batch or fed-batch culture, where the culture is terminated after sufficient expression of the recombinant protein (e.g., an antibody), and then the expressed protein (e.g., an antibody) is harvested. Alternatively, as another non-limiting example, the cells may be grown in batch-refeed, where the culture is not terminated and fresh nutrients and other components are added to the culture periodically or continuously, while the expressed recombinant protein (e.g., an antibody) is harvested periodically or continuously. Other suitable methods (e.g., spin tube culture) are known in the art and can be used to carry out the methods described herein.

[0044] In some embodiments, the cell culture is a fed-batch culture. As used herein, the term "fed-batch culture" refers to a method of culturing cells in which additional components are provided to the culture one or more times after the start of the culture process. Such provided components typically include the nutrient components of the cells that have been depleted during the culture process. Fed-batch culture typically stops at some point and the cells and / or components in the medium are harvested and optionally purified. In some embodiments, the fed-batch culture includes a basal medium supplemented with a feed medium.

[0045] Alternatively, the cells can be grown in a perfusion process. In a perfusion process, the cells are retained within the bioreactor while continuously removing cell waste and depleted medium. The rate at which fresh medium is added is approximately the same as the rate at which the emptied medium is removed from the system. Removal of the spent medium can be performed by alternating tangential flow, standard tangential flow filtration, hollow fiber filtration, etc.

[0046] The cells may be grown in any convenient volume selected by the practitioner. For example, the cells may be grown in a small-scale reaction vessel having a volume in the range of a few milliliters to a few liters. Alternatively, the cells may be grown in a large-scale, commercially available bioreactor having a volume of at least approximately 1 liter to 10, 50, 100, 250, 500, 1000, 2500, 5000, 8000, 10000, 12000, 15000, 20000 or 25000 liters or more, or any volume range therebetween.

[0047] The temperature of the cell culture is selected primarily based on the range of temperatures in which the cell culture can continue to survive and the range in which a high level of the desired product (e.g., a recombinant protein) is produced. Generally, most mammalian cells grow well within the range of about 25°C to 42°C and can produce the desired product (e.g., a recombinant protein), but the methods described herein are not limited to these temperatures. Certain mammalian cells grow well within the range of about 35°C to 40°C and can produce the desired product (e.g., a recombinant protein or antibody). In certain embodiments, the cell culture is grown at a temperature of 20-45°C one or more times during the cell culture process. One of ordinary skill in the art will be able to select the appropriate temperature(s) for growing the cells depending on the specific needs of the cells and the specific production requirements of the practitioner. The cells may be grown for any period of time depending on the needs of the practitioner and the requirements of the cells themselves. In one embodiment, the cells are grown at 37°C. In other embodiments, the cells are grown at 36.5°C.

[0048] In some embodiments, the cells are grown for a longer or shorter time during the initial growth phase (or growth phase), depending on the needs of the practitioner and the requirements of the cells themselves. In some embodiments, the cells are grown for a time sufficient to achieve a predetermined cell density. In some embodiments, the cells are grown for a time sufficient to achieve a cell density that is a predetermined percentage of the maximum cell density that the cells would ultimately reach if grown without interference. For example, the cells can be grown for a period sufficient to achieve a desired viable cell density of 1-99% of the maximum cell density. In some embodiments, the cells are grown until the cell density does not increase by more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% per day of culture. In some embodiments, the cells are grown until the cell density no longer increases by more than 5% per day of culture.

[0049] In some embodiments, the cells can be grown for a defined period. For example, depending on the starting concentration of the cell culture, the temperature at which the cells grow, and the intrinsic growth rate of the cells, the cells can be grown for 0-20 days or more, specifically 4-10 days. In some cases, the cells can be grown for more than one month. The practitioner can select the duration of the initial growth phase according to the requirements of protein production and the needs of the cells themselves.

[0050] The cell culture can be stirred or shaken during the initial culture phase to increase the oxygenation and dispersion of nutrients to the cells. Those skilled in the art will understand that it may be beneficial to control or regulate certain internal conditions of the bioreactor during the initial growth phase, including but not limited to pH, temperature, oxygenation, etc.

[0051] At the end of the initial growth phase, at least one of the culture conditions can be shifted to apply a second set of culture conditions to cause a metabolic shift in the culture. The metabolic shift can be achieved, for example, by a change in the temperature, dissolved oxygen level, pH, osmotic pressure, or chemical inducer level of the cell culture. In one non-limiting embodiment, the culture conditions are shifted by shifting the temperature of the culture. However, as is known in the art, a temperature shift is not the only mechanism by which an appropriate metabolic shift can be achieved. For example, such a metabolic shift can also be achieved by shifting other culture conditions including, but not limited to, pH, dissolved oxygen, osmotic pressure, and sodium butyrate levels. The timing of the culture shift is determined by the practitioner based on the requirements of protein production or the needs of the cells themselves.

[0052] When shifting the temperature of the culture, the temperature shift is relatively gradual. For example, it may take several hours or days for the temperature change to be completed. Alternatively, the temperature shift may be relatively rapid. For example, the temperature change may be completed within a few hours. Considering appropriate production and control equipment such as that which is standard in the commercial large-scale production of polypeptides or proteins, the temperature change can be completed in less than one hour.

[0053] In some embodiments, once the conditions of the cell culture are shifted as described above, the cell culture is maintained for the next production phase under a second set of culture conditions that are useful for the survival and viability of the cell culture and appropriate for the expression of the desired polypeptide or protein at a commercially appropriate level.

[0054] As described above, the culture can be shifted by shifting one or more of a number of culture conditions including, but not limited to, temperature, dissolved oxygen, pH, osmotic pressure, and sodium butyrate level. In some embodiments, the temperature of the culture is shifted. According to this embodiment, during the subsequent production phase, the culture is maintained at a temperature or temperature range lower than the temperature or temperature range of the initial growth phase. As described above, multiple individual temperature shifts can be used to increase cell density or viability, or to increase the expression of a recombinant protein.

[0055] Cells can express a recombinant protein, gene product, or cell product. In some embodiments, the cells express a recombinant protein and the cell culture method includes a growth phase and a production phase. The methods described herein can be applied during the growth phase, the production phase, or both.

[0056] The methods described herein can be used to improve cell growth of high-density cells in high-density cell culture. As used herein, high cell density refers to a cell density greater than 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL or 5×10 8 cells / mL, preferably greater than 1×10 7 cells / mL, more preferably greater than 5×10 7 cells / mL.

[0057] In some embodiments, cell growth is determined by viable cell density (VCD), maximum viable cell density, or integrated viable cell count (IVCC). In some embodiments, cell growth is determined by maximum viable cell density. As used herein, the term "viable cell density" refers to the number of cells present in a given volume of culture medium. Viable cell density can be measured by any method known to those skilled in the art. Preferably, viable cell density is measured using an automated cell counter such as Bioprofile Flex®. As used herein, the term "maximum cell density" refers to the maximum cell density achieved during cell culture. As used herein, the term "cell viability" refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. For example, to determine cell viability, a dye (e.g., trypan blue) that cannot pass through the membranes of living cells but can pass through the disrupted membranes of dead or dying cells can be used.

[0058] As used herein, the term "Integrated viable cell count (IVCC)" refers to the area under the viable cell density (VCD) curve. IVCC can be calculated using the following formula: IVCC t+1 =IVCC t +(VCD t +VCD t+1 ) * (Δt) / 2 where Δt is the time difference between time points t and t + 1. IVCC t=0 can be considered negligible. VCD t and VCD t+1 are the viable cell densities at time points t and t + 1.

[0059] As used herein, the term "titer" refers to, for example, the total amount of recombinant expressed protein produced by a cell culture in a given volume of culture medium. Titer is typically expressed in grams of protein per liter of culture medium.

[0060] In some embodiments, cell growth increases by at least 5%, 10%, 15%, 20% or 25% compared to a control culture. The control culture can be identical to the above-described cultures, except that it is not cultured using step (ii). In some embodiments, cell growth increases by at least 10% compared to a control culture. In some embodiments, cell growth increases by at least 20% compared to a control culture.

[0061] In some embodiments, productivity is determined by titer and / or volumetric productivity. As used herein, the term "titer" refers to, for example, the total amount of recombinant expressed protein produced by cell culture in a given volume of medium. Titer is typically expressed in grams of protein per liter of medium. In some embodiments, productivity is determined by titer. In some embodiments, productivity increases by at least 5%, 10%, 15%, 20% or 25% compared to a control culture. In some embodiments, productivity increases by at least 10% compared to a control culture. In some embodiments, productivity increases by at least 20% compared to a control culture.

[0062] In some embodiments, the maximum cell density of the cell culture is 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL or 5×10 8 cells / mL or greater. In some embodiments, the maximum cell density of the cell culture is greater than 5×10 6 cells / mL. In some embodiments, the maximum cell density of the cell culture is greater than 1×10 8 cells / mL.

[0063] As used herein, the terms “medium,” “cell culture medium,” and “culture medium” refer to a solution containing nutrients that nourish growing mammalian cells. Typically, such a solution provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cells for minimal growth and / or survival. Such a solution may also contain hormones and / or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, phosphate, etc.), buffers, vitamins, nucleosides or nucleotides, trace elements (usually inorganic compounds present at very low final concentrations), inorganic compounds present at high final concentrations (such as iron), amino acids, lipids, and / or auxiliary components that enhance growth and / or survival beyond a minimal rate, including but not limited to glucose or other energy sources. In some embodiments, the medium is advantageously formulated to have a pH and salt concentration optimal for cell survival and growth. In some embodiments, the medium is a feed medium that is added after the initiation of cell culture.

[0064] In accordance with the methods described herein, a wide variety of mammalian growth media can be used. In some embodiments, the cells can be grown in one of a variety of chemically defined media in which the components of the medium are known and controlled. In some embodiments, the cells can be grown in a complex medium in which not all of the components of the medium are known and / or controlled.

[0065] Chemically defined growth media for mammalian cell culture have been widely developed and published over the past few decades. All components of the defined media are well characterized, and thus the defined media do not contain complex additives such as serum or hydrolysates. Initial media formulations were developed to allow cell growth and maintenance with little or no regard for protein production. More recently, media formulations have been developed with the explicit aim of supporting highly productive recombinant protein-producing cell cultures. Such media generally contain large amounts of nutrients, particularly amino acids, to support cell growth and / or maintenance at high density. These media may be modified by those skilled in the art for use in the methods described herein, if desired.

[0066] Since not all components of complex media are well characterized, complex media may contain, among other things, simple and / or complex carbon sources, simple and / or complex nitrogen sources, and additives such as serum. In some embodiments, the complex media contain additives such as hydrolysates in addition to other components of the defined media as described herein.

[0067] In some embodiments, defined media typically contain about 50 chemicals at known concentrations in water. Most of them also contain one or more well-characterized proteins such as insulin, IGF-1, transferrin or BSA, while others do not require a protein component and are thus called protein-free media. The typical chemical components of media are classified into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and other categories that do not fall neatly into other classifications.

[0068] Optional auxiliary components may be added to the cell culture medium. As used herein, the term "auxiliary components" refers to components that enhance growth and / or survival above a minimal rate, including but not limited to hormones and / or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, phosphate, etc.), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, and / or glucose or other energy sources. In some embodiments, the auxiliary components can be added to the initial cell culture. In some embodiments, supplementary components may be added after the start of cell culture.

[0069] Typically, trace elements refer to various inorganic salts contained at levels below micromolar. For example, commonly included trace elements are zinc, selenium, copper, etc. In some embodiments, iron (ferrous or ferric salts) can be included as a trace element in the initial cell culture medium at micromolar concentrations. Manganese is also often included as a divalent cation (MnCl 2 or MnSO 4 ) in the trace elements in the range of nanomolar to micromolar concentrations. Usually, many less common trace elements are added at nanomolar concentrations.

[0070] In some embodiments, the medium used in the method is suitable for supporting a high cell density in the cell culture, such as 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL or 5×10 8 cells / mL. In some embodiments, the cell culture is a mammalian cell fed-batch culture, preferably a CHO cell fed-batch culture.

[0071] Often, cells are selected or engineered to produce high levels of a desired product (e.g., a recombinant protein or antibody). Often, cells are engineered to produce high levels of a recombinant protein, for example, by introduction of a gene encoding the protein of interest and / or introduction of gene regulatory elements (whether endogenous or introduced) that regulate the expression of that gene.

[0072] Certain proteins can have a detrimental effect on cell growth, cell viability, or other characteristics of the cell that ultimately limit production of the protein of interest in some way. Even among a population of a particular type of cell engineered to express a particular protein, there is variability within the cell population such that certain individual cells grow better, produce more of the protein of interest, or produce a protein with a higher activity level (e.g., enzyme activity). In certain embodiments, cell lines are empirically selected by the practitioner to ensure growth under the particular conditions selected for culturing the cells. In some embodiments, individual cells engineered to express a particular protein are selected for large-scale production based on cell growth, final cell density, cell viability, titer of the expressed protein, or any combination thereof, or any other condition deemed important by the practitioner.

[0073] Any protein that can be expressed in a host cell can be produced according to the methods described herein. As used herein, the term "host cell" refers to a cell engineered to produce a protein of interest as described herein. The protein can be expressed from a gene endogenous to the cell or from a heterologous gene introduced into the cell. The protein can be a protein that occurs in nature or can have a genetically engineered or selected sequence.

[0074] Proteins that may desirably be expressed are often selected based on interesting or useful biological or chemical activities. For example, the method can be used to express any enzyme, receptor, antibody, hormone, regulatory factor, antigen, binding agent, etc. that is pharmaceutically or commercially relevant. In some embodiments, the protein expressed by the cells in culture is selected from an antibody or fragment thereof, nanobody, single-domain antibody, glycoprotein, therapeutic protein, growth factor, coagulation factor, cytokine, fusion protein, pharmaceutical drug substance, vaccine, enzyme, or Small Modular ImmunoPharmaceuticals™ (SMIPs). Those skilled in the art will understand any protein that can be expressed and any protein for which it is possible to select a specific protein produced based on the particular needs of those skilled in the art.

[0075] According to the method of the present invention, the production of antibodies is of particular interest. An antibody is a protein that has the ability to specifically bind to a particular antigen. Any antibody that can be expressed in a host cell can be produced. In some embodiments, the antibody expressed is a monoclonal antibody.

[0076] In some embodiments, the monoclonal antibody is a chimeric antibody. A chimeric antibody contains amino acid fragments from multiple organisms. A chimeric antibody molecule can, for example, contain an antigen-binding domain from an antibody of mouse, rat, or other species having a human constant region.

[0077] In some embodiments, the monoclonal antibody is a human antibody induced, for example, through the use of ribosome display or phage display libraries, or the use of xenografts in which the native antibody genes are inactivated and functionally replaced with human antibody genes while other components of the native immune system remain intact.

[0078] In some embodiments, the monoclonal antibody is a humanized antibody. A humanized antibody is a chimeric antibody in which most of the amino acid residues are derived from human antibodies, and thus minimizes any potential immune response when delivered to a human subject. In a humanized antibody, the amino acid residues of the complementarity determining regions are at least partially replaced with residues from a non-human species that confer the desired antigen specificity or affinity. Such modified immunoglobulin molecules can be made by any of several techniques known in the art.

[0079] In some embodiments, the above monoclonal antibody, chimeric antibody, or humanized antibody may contain amino acid residues that do not naturally exist in any antibody of any species. These foreign residues can be utilized, for example, to confer novel or modified specificity, affinity, or effector function to the monoclonal antibody, chimeric antibody, or humanized antibody. In some embodiments, the above antibodies can be conjugated to drugs for systemic drug therapy such as toxins, low molecular weight cytotoxic drugs, biological response modifiers, and radionuclides.

[0080] Generally, a nucleic acid molecule introduced into a cell encodes a protein that is desired to be expressed. Alternatively, the nucleic acid molecule can encode a gene product that induces the expression of a desired protein by the cell. For example, the introduced genetic material can encode a transcription factor that activates the transcription of an endogenous or heterologous protein. Alternatively or concomitantly, the introduced nucleic acid molecule can increase the translation or stability of a protein expressed by the cell. Methods suitable for introducing sufficient nucleic acid to achieve expression of the protein of interest into mammalian host cells are known in the art. Common methods for introducing genetic material into mammalian cells include the calcium phosphate precipitation method or the Lipofectamine™ method.

[0081] In some embodiments, the nucleic acid introduced is in the form of a naked nucleic acid molecule. For example, the nucleic acid molecule introduced into a cell can consist only of a nucleic acid encoding a protein and the necessary genetic control elements. Alternatively, a nucleic acid encoding a protein (including the necessary regulatory elements) may be contained within a plasmid vector. Non-limiting representative examples of suitable vectors for protein expression in mammalian cells include pcDNA1; pCD; pMCIneo poly-A; baculovirus vectors such as pAC373 or pAC610; CDM8; and pMT2PC. In some embodiments, the nucleic acid molecule introduced into a cell is contained within a viral vector. For example, a nucleic acid encoding a protein can be inserted into a viral genome (or a partial viral genome). Regulatory elements that direct protein expression can be included with the nucleic acid inserted into the viral genome (i.e., linked to the gene inserted into the viral genome) or can be provided by the viral genome itself.

[0082] Naked DNA can be introduced into cells by forming a precipitate containing DNA and calcium phosphate. Alternatively, naked DNA can be introduced into cells by forming a mixture of DNA and DEAE-dextran and incubating this mixture with the cells, or by incubating the cells and DNA together in a suitable buffer and subjecting the cells to high voltage electrical pulses (e.g., by electroporation). A further method for introducing naked DNA into cells is by mixing DNA with a liposome suspension containing a cationic lipid. The DNA / liposome complex is then incubated with the cells. Naked DNA can also be directly injected into cells, for example, by microinjection. Alternatively, naked DNA can be introduced into cells by complexing the DNA with a cation such as polylysine that is bound to a ligand for a cell surface receptor. When the DNA-ligand complex binds to the receptor, uptake of the DNA by receptor-mediated endocytosis is promoted.

[0083] Defective retroviruses have been well-characterized for use in gene transfer for gene therapy purposes. Nucleic acids encoding a protein of interest can be inserted into the retroviral genome to construct recombinant retroviruses. Furthermore, a portion of the retroviral genome can be removed to render the retrovirus replication-deficient. Such replication-deficient retroviruses are then packaged into virions that can be used to infect target cells via the use of helper viruses by standard techniques. For example, the genome of an adenovirus can be engineered to encode and express a protein of interest but be inactivated with respect to its ability to replicate in the normal lytic viral life cycle. Exemplary adenoviral vectors derived from the adenovirus strain Ad5 type d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known to those of skill in the art. Adeno-associated virus (AAV) is a naturally occurring defective virus that requires another virus, such as an adenovirus or herpesvirus, as a helper virus for efficient replication and a productive life cycle.

[0084] If the method used to introduce a nucleic acid molecule into a cell population results in modification of the majority of the cells and efficient expression of the protein by the cells, the modified cell population can be used without the need for further isolation or subcloning of individual cells within the population. That is, there can be sufficient production of the protein by the cell population such that the population can be used immediately to seed a cell culture for protein production without the need for further cell isolation. Alternatively, it may be desirable to isolate and expand a homogeneous population of cells from a small number of cells or a single cell that efficiently produce the protein.

[0085] Instead of introducing a nucleic acid molecule encoding a protein of interest into a cell, the introduced nucleic acid may encode another polypeptide or protein that induces or increases the expression level of a protein endogenously produced by the cell. For example, a cell may be able to express a particular protein, but may not be able to do so without further manipulation of the cell. Similarly, a cell may express an insufficient amount of a protein for a desired purpose. Thus, an agent that stimulates the expression of the protein of interest can be used to induce or increase the expression of that protein by the cell. For example, the introduced nucleic acid molecule may encode a transcription factor that activates or upregulates the transcription of the protein of interest. When such a transcription factor is expressed, it in turn results in the expression, or stronger expression, of the protein of interest.

[0086] Generally, typically, it may be desirable to isolate and / or purify a recombinant protein, gene product, or cell product expressed according to the methods described herein. In certain embodiments, the expressed protein is secreted into the medium and thus cells and other solids can be removed as a first step in the purification process, for example, by centrifugation or filtration.

[0087] Alternatively, the expressed protein may be bound to the surface of the host cell. For example, the medium can be removed and the host cells expressing the protein can be lysed as a first step in the purification process. Lysis of mammalian host cells can be accomplished by any number of means well known to those of skill in the art, including physical disruption with glass beads and exposure to high pH conditions.

[0088] The expressed protein can be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation, and / or any other available technique for protein purification.

[0089] In certain embodiments, the produced polypeptide or protein will have pharmacological activity and will be useful in the preparation of pharmaceuticals. Proteins and peptides can be formulated for delivery by any available route including, but not limited to, parenteral (e.g., intravenous administration), intradermal, subcutaneous, oral, nasal, bronchial, ocular, transdermal (topical), transmucosal, rectal, and vaginal routes. Pharmaceutical compositions typically include a purified polypeptide or protein expressed from a mammalian cell line, a delivery agent in combination with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0090] The invention is further illustrated by the following non-limiting examples.

Example

[0091] Example 1: Identification of Inhibitors The entire CHO metabolome was screened using HILIC liquid chromatography column Orbitrap™ mass spectrometry (LC-MS). Over 30,000 features were analyzed and over 1,000 features were accumulated through the cell culture. The inhibitory effects of the top 20 features were analyzed and tested biologically. The metabolites structurally confirmed by LC-MS are shown in Table 1. The concentrations at the end of the culture were also measured in different ways using LC-MS.

[0092]

Table 1

[0093] Example 2: Standard Bioprocess and Biological Confirmation of Metabolites CHO K-1 Industrial Standard Batch Process: The NIH CHO-K1 cell line was cultured for 6 days, and 6 mM glutamine supplement was added on the inoculation day. The working volume was 30 mL in a 125 mL shaking flask, and the inoculation cell density was 500,000 cells / mL. The parameters of the shaking incubator were 125 RPM and 5% CO 2 is.

[0094] CHO GS Industrial Standard Batch Process: The CHOZn® cell line was cultured for 6 days. The working volume was 30 mL in a 125 mL shaking flask, and the inoculation cell density was 500,000 cells / mL. The parameters of the shaking incubator were 125 RPM and 5% CO 2 is.

[0095] HEK293 Industrial Standard Batch Process: The MBL HEK293 cell line was cultured for 6 days. The working volume was 30 mL in a 125 mL shaking flask, the inoculation cell density was 500,000 cells / mL, and 6 mM glutamine supplement was added on the inoculation day. The parameters of the shaking incubator were 125 RPM and 5% CO 2 is.

[0096] The cells were exposed to the identified inhibitory metabolites in Table 1 at their concentrations at the end of their culture in batch culture for 6 days. AA, ICA, CMP, and the mixture condition showed lower cell density than the control group (Figure 3). All inhibitory metabolites decreased antibody productivity (Figure 4).

[0097] Industrial Standard Fed-Batch Process: The CHO-K1 cell line was cultured in the corresponding medium. 6 mM glutamine supplement was added on the inoculation day and fed for 14 days. The working volume was 30 mL in a 125 mL shaking flask, and the inoculation cell density was 500,000 cells / mL. The parameters of the shaking incubator were 125 RPM and 5% CO 2 is. The feeding strategy was 10% corresponding feeding daily from day 3 to day 13.

[0098] In a standard industrial fed-batch process, cells were exposed to inhibitors for a long period. This experiment mimicked the therapeutic antibody production process and spiked the inhibitor at the cell concentration at the end of the culture. ICA was identified as a known inhibitor and used as a positive inhibitor control. The mixing conditions excluded ICA and included AA, HICA, CMP, MSA, TRI, and NAP. This process was continued for 14 days. From the 6th day, the cell density under the inhibitor conditions was lower than that of the negative control batch. The results are shown in Figure 3. Compared with the control batch, CMP, ICA, TRI, and HICA showed a stronger growth inhibition effect, resulting in a 40 - 50% VCD decrease at the peak VCD on the 9th day.

[0099] Example 3 shows the effects on metabolite productivity and quality. The IgG titer was measured by HPLC using a Protein A column. The quality of the product was quantified by the IgG glycan profile measured by HPLC using a Glycan Column.

[0100] All seven metabolites showed an inhibitory effect on productivity and product quality. HICA, CMP, ICA, and TRI showed strong inhibition on productivity, with a productivity decrease of more than 30% compared to the control (Figure 4). The quality of the product is an important parameter for bioprocess and pharmaceutical approval, and the glycan profile is one of the most important critical quality attributes (CQAs). AA, HICA, CMP, MSA, and ICA showed a higher impact on G1F formation (Figure 5). AA completely inhibited G2F formation, and for G2F formation, all the other six inhibitors showed a significant impact (Figure 6). The repeated spike experiments showed consistent effects on the glycan profile (Figures 7, 8, and 9).

[0101] Conclusion More than 30,000 features were captured by the LC-MS metabolomics study described in this specification, and the top 20 inhibitor candidates with high reliability in the identification matching were selected by multivariate data analysis and pathway analysis. The top candidates were experimentally verified by spiking them into batch cultures, and 11 out of 15 of them showed an inhibitory effect on cell growth. Eight metabolic by-products were structurally and biologically verified. Among these, six metabolites are novel inhibitory by-products of cell growth and / or productivity, and four of them are CHO metabolites identified for the first time (see Table 2).

[0102]

Table 2

[0103] Example 4: Accumulation of Inhibitory Metabolites in Fed-Batch Culture The fed-batch conditions were the same as the CHO K-1 industrial standard fed-batch culture process of Example 2. The batch process conditions were the same as the CHO K-1 industrial standard batch process of Example 2.

[0104] Metabolites were identified by LC-ESI-MS. Table 3 shows the identified inhibitory metabolites accumulated in the process and the accumulations at 6 days in the batch process and at 5, 8, and 14 days in the fed-batch process.

[0105]

Table 3

[0106] This example shows the inhibitor concentrations in batch culture and fed-batch culture. It was concluded that inhibitory metabolites accumulated in the main bioprocess. Example 5: Accumulation of Inhibitory Metabolites in CHO K1, CHO GS, and HEK293 in Batch Culture The batch process conditions were the same as those of the CHO K-1, CHO GS, and HEK293 industrial standard batch processes of Example 2. Metabolites were identified by LC-ESI-MS. Table 4 shows the identified inhibitory metabolites accumulated in the process and their accumulation after 6 days in the batch process. Table 4 shows that six inhibitory metabolites are present in different mammalian cell culture systems and accumulate in batch culture.

[0107]

Table 4

[0108] From the final accumulation in batch culture, it was concluded that inhibitory metabolites are widely present in mammalian cell lines. Example 6: Process Development to Mitigate the Accumulation of Inhibitory Metabolites Transfection of the CHO K1 cell line with regulatory genes for the production of inhibitory metabolites in batch culture mitigates the growth of inhibitory metabolites.

[0109] The purpose of transfection is to overexpress the regulatory genes / enzymes of inhibitory metabolites. The downstream enzyme-coding genes were knocked into the CHO cell line through a plasmid constructed with Got1, Hoga1, Cat, and Slc35a1 on the pcDNA3.1(+) vector (V79020, ThermoFisher Scientific). The plasmid was amplified using 5α competent E. coli (C2987I, New England BioLabs). The plasmid was transfected into the mammalian cell line using polyethyleneimine (408727, Millipore Sigma), and the concentration of inhibitory metabolites was mitigated by overexpressing the regulatory genes of inhibitory metabolites. The transfection was the same as the CHO K1 industrial standard batch process except that the initial inoculation cell density was 1 million cells / mL.

[0110] In Table 5 and Figure 11, the control is CHO without transfection of any gene Shows the K1 batch cell culture control conditions; T1 shows the cell growth profile of CHO K1 cell line transfected with Got1; T2 shows the Got1 and Hoga1 transfection; and T3 shows the transfection of Got1, Hoga1, Cat, Slc35a1. As shown in comparison with the control cell culture in Table 5 and Figure 11, with the overexpression of inhibitory metabolite regulatory genes in the CHO K1 cell line, the concentrations of all inhibitory metabolites decrease and the growth profile is improved.

[0111]

Table 5

[0112] From this data, it was concluded that the inhibitor concentration decreased after transfection of the inhibitory metabolite regulatory gene into the CHO cell line. The use of the terms "a," "an," and "the" and similar references (especially in the context of the following claims) shall be construed to include both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context. The terms first, second, etc. used herein are not meant to indicate any particular order but are for convenience only to refer to a plurality, for example, of layers. The terms "comprising," "having," "including," and "containing" shall be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise stated. The recitation of a range of values is intended solely as a concise method of referring individually to each value within the range and each individual value is incorporated herein as if it were individually recited herein. All endpoints of the ranges are included within the range and may be combined independently. All methods described herein can be performed in a suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any examples, or exemplary language (e.g., "such as") is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention as used herein.

[0113] Although the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes can be made without departing from the scope of the present invention, and equivalents can be substituted for its elements. Furthermore, many modifications can be made to adapt a particular situation or material to the teachings of the present invention without departing from the essential scope thereof. Accordingly, it is intended that the present invention not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out the present invention, but that the present invention will include all embodiments within the scope of the appended claims. Any combination of the above elements in all possible variations thereof is included by the present invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. In a method for culturing cells, the method comprises: (i) culturing the cells in a cell culture medium; (ii) maintaining at least one metabolite selected from aconitic acid (AA), leucine acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI) and N-acetylputresinium (NAP) in the cell culture medium at a sub-inhibitory concentration for the at least one metabolite; A method comprising:

2. the metabolite is AA and the inhibitory concentration is less than 100 μM, 440 μM, 880 μM, 3 mM, 5 mM, or 10 mM; said metabolite is HICA and said inhibitory concentration is less than 10 μM, 23.5 μM, 47 μM, 100 μM, 1 mM or 3 mM; said metabolite is CMP and said inhibitory concentration is 5 μM, 10 μM, 20 μM, 100 μM, 500 μM or 1 mM; the metabolite is MSA and the inhibitory concentration is less than 1 μM, 3.75 μM, 7.5 μM, 100 μM, 1 mM, or 3 mM; the metabolite is TRI and the inhibitory concentration is less than 0.1 μM, 0.35 μM, 0.7 μM, 100 μM, 1 mM, or 3 mM; or 2. The method of claim 1, wherein the metabolite is NAP and the inhibitory concentration is less than 0.1 μM, 0.3 μM, 0.6 μM, 100 μM, 1 mM or 3 mM.

3. 2. The method of claim 1, wherein the concentration of the at least one metabolite is measured using nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, high performance / ultra performance liquid chromatography (H / UPLC), liquid chromatography mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS) techniques, or a combination thereof.

4. 2. The method of claim 1, wherein step (ii) comprises measuring a concentration of the at least one metabolite, and if the measured concentration of the at least one metabolite exceeds a predetermined value, decreasing a concentration of a precursor of the at least one metabolite in the cell culture medium by reducing an amount of the precursor provided to the cells.

5. When the metabolite is AA, the precursor is glutamine, glucose, arginine, asparagine, or a combination thereof; When the metabolite is HICA, the precursor is leucine, isoleucine, or a combination thereof; When the metabolite is CMP, the precursor is glutamine, arginine, aspartic acid, or a combination thereof; When the metabolite is MSA, the precursor is lysine, isoleucine, serine, glucose, glutamine or a combination thereof; When the metabolite is TRI, the precursor is aspartate, tryptophan, glutamine, or a combination thereof; Where the metabolite is NAP, the precursor is arginine, proline, aspartic acid, glutamine, asparagine or a combination thereof; or The method according to claim 4, which is a combination thereof.

6. 2. The method of claim 1, wherein step (ii) comprises measuring a concentration of the at least one metabolite, and modulating expression of an enzyme to reduce synthesis of the metabolite if the measured concentration of the at least one metabolite exceeds a predetermined value.

7. the metabolite is AA and the enzyme is ADI1, HOGA1, TAD1, or a combination thereof; the metabolite is HICA and the enzyme is GOT1, D-HicDH, MMUT, AUH, HMGCL, HADHA / B, or a combination thereof; the metabolite is CMP and the enzyme is UCK1 / 2, NT5, CMAS, CMPK1, DDYD, CDA, SLC35A1, RRM1, HOGA1, or a combination thereof; the metabolite is MSA and the enzyme is GOT1, ETHE1, AMT, HADHA / B, MMUT, or a combination thereof; the metabolite is TRI and the enzyme is NADSYN1, NNMT, CAT, NMNAT1, SULT4A1, or a combination thereof; The metabolite is NAP and the enzyme is SAT1 / 2, HOGA1, AMD1, ODC1, GOT1, MAOB, or a combination thereof; or The method of claim 6, which is a combination thereof.

8. The method of claim 6, wherein regulating enzyme expression to reduce synthesis of the metabolite comprises adding an inhibitory nucleic acid that inhibits expression of a gene encoding the enzyme.

9. 7. The method of claim 6, wherein regulating enzyme expression to reduce synthesis of the metabolite comprises adding a recombinant DNA molecule to overexpress a regulatory enzyme, the regulatory enzyme being a downstream enzyme.

10. 2. The method of claim 1, wherein step (ii) comprises measuring a concentration of said at least one metabolite, and modulating an activity of an enzyme to reduce synthesis of said metabolite if said measured concentration of said at least one metabolite exceeds a predetermined value.

11. the metabolite is AA and the enzyme is ADI1, HOGA1, TAD1, or a combination thereof; the metabolite is HICA and the enzyme is D-HicDH, MMUT, AUH, HMGCL, HADHA / B, or a combination thereof; the metabolite is CMP and the enzyme is UCK1 / 2, NT5, CMAS, CMPK1, DDYD, CDA, SLC35A1, RRM1, HOGA1, or a combination thereof; the metabolite is MSA and the enzyme is ETHE1, AMT, HADHA / B, MMUT, or a combination thereof; the metabolite is TRI and the enzyme is NADSYN1, NNMT, CAT, NMNAT1, SULT4A1, or a combination thereof; The metabolite is NAP and the enzyme is SAT1 / 2, HOGA1, AMD1, ODC1, GOT1, MAOB, or a combination thereof; or The method of claim 10 or a combination thereof.

12. The method of claim 10, wherein modulating the enzyme activity to reduce synthesis of the metabolite comprises adding an inhibitor of the enzyme activity to the culture.

13. The method of claim 10, wherein modulating enzyme activity to reduce synthesis of the metabolite comprises adding a glycolytic pathway activator to the culture.

14. 2. The method of claim 1, wherein for the at least one metabolite, maintaining the at least one metabolite in the cell culture medium below an inhibitory concentration comprises controlling temperature, dissolved oxygen levels, pH, or a combination thereof.

15. The method of any one of claims 1 to 14, wherein the cell is a CHO cell, a HEK293 cell, a HT-1080 cell, a genetically engineered T cell, or a genetically engineered natural killer cell.

16. The method according to any one of claims 1 to 15, wherein the cell culture is a batch culture, a fed-batch culture, or a perfusion culture.

17. The method of any one of claims 1 to 16, wherein the cells express a recombinant protein, gene product, or cell product.

18. 20. The method of claim 17, further comprising obtaining and purifying the recombinant protein, gene product, or cell product.

19. 18. The method of claim 17, wherein the recombinant protein is a monoclonal antibody.

20. 20. The method of any one of claims 1 to 19, wherein cell proliferation and / or productivity is increased compared to a control culture, said control culture being identical to the culture of claim 1 except that it is not cultured using step (ii).

21. 21. The method of claim 20, wherein the cell proliferation is determined by maximum viable cell density and is increased by at least 5% compared to the control culture.

22. The maximum viable cell density of the cell culture was 1×10 ６ cells / mL, 5 x 10 ６ cells / mL, 1 x 10 ７ cells / mL, 5 x 10 ７ cells / mL, 1 x 10 ８ cells / mL or 5 x 10 ８ The method of any one of claims 1 to 21, wherein the concentration is greater than cells / mL.

23. The method according to any one of claims 1 to 22, wherein the cell culture method comprises a growth phase and a production phase, and step (ii) is applied during the growth phase.

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