Cell culture method

Abstract

The cell culture method includes (i) culturing cells in a cell culture medium; and (ii) maintaining at least one metabolite in the cell culture medium at a below inhibitory concentration of the at least one metabolite, wherein the at least one metabolite is aconitic acid (AA), leucic acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI), N-acetylputresinium (NAP), or a combination thereof, and the enzyme includes ADH5, BCAT1, CAT, GOT1, HADHB, HOGA1, SLC35A1, or a combination thereof.

Description

[Technical field]

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

[0002] Mammalian cells, such as Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cell lines, are used as hosts to manufacture therapeutic biologics in the biopharmaceutical industry. In the process of antibody production, for example, CHO cells exhibit inefficient and poorly controlled metabolism, as they tend to generate waste metabolites using much of the available nutrients instead of the amount required for their growth. In the later stages of the bioprocess, the cells stop growing and stop producing antibodies, even though the nutrient levels are sufficient to support growth and protein production. Metabolites accumulated during the process are one of the major rate-limiting factors in cell growth and antibody production. To control metabolism and increase the productivity of mammalian cells, it is important to identify inhibitory waste metabolites and study their pathways. Summary of the Invention [Problem to be solved by the invention]

[0003] There is a need to identify inhibitory metabolites and develop control strategies. [Means for solving the problem]

[0004] In one aspect, a method of cell culture comprises: (i) culturing cells in a cell culture medium; and (ii) maintaining in the cell culture medium a sub-inhibitory concentration of at least one metabolite, wherein the at least one metabolite is aconitic acid (AA), leucic acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI), and N-acetylputrescinium (NAP), or a combination thereof, and the enzyme comprises ADH5, BCAT1, CAT, GOT1, HADHB, HOGA1, SLC35A1, or a combination thereof. [Brief description of the drawings]

[0005] [Figure 1] Integral viable cell density is shown as the accumulation of cells in batch culture. Mixture conditions exclude ICA and include AA, HICA, CMP, MSA, TRI, and NAP. [Diagram 2] Figure 2 shows antibody production inhibited by inhibitory metabolites. Productivity is shown as a normalized scale. Mixing conditions were the same as in Figure 1. [Diagram 3] 1 shows viable cell density plots for fed-batch spiking studies. [Figure 4] HICA, CMP, ICA and TRI (30-40% reduction in titer compared to control) were more potent inhibitors of productivity. Values ​​are titer values ​​at harvest (day 14) measured by HPLC using triplicate standard curves. [Diagram 5] The effect of glycosylation levels on product quality for G1F, one of the glycoprofile species found in IgG antibody drugs, is shown in Figure 1. Compared to the control group (column 9), AA, HICA, CMP, MSA, and ICA had a greater effect on G1F formation. [Figure 6]The effect of product quality on G2F, one of the glycoprofile species found in IgG antibody drugs, is shown in the figure, showing glycosylation levels. Compared to the control group (column 9), AA completely inhibited G2F formation, and all other six inhibitors had a significant effect on G2F formation. [Figure 7] Liquid chromatogram of the control culture on day 14. G0F, G1F and G2F glycoprofile species were observed during the bioprocess. [Figure 8] Liquid chromatograms of AA spiked cultures on day 14. Only G0F and G1F peaks were observed; G2F was not formed during the bioprocess. [Figure 9] Liquid chromatograms of AA spiked cultures on day 14 are shown. [Figure 10] Here is an example of a NAP control strategy. This is the N-acetylglutaric acid (NAP) metabolic pathway in CHO cells. Control strategy 1: Reduce the concentration of proline and arginine in the medium to reduce the production of NAP. In the current CHO cell bioprocess, overfeeding (too high nutrients) is very common, which will affect the production and quality of biomanufacturing. Control strategy 2: Reduce the expression level of the enzyme (SAT1 / 2), such as by introducing siRNA. The reduced expression level of the enzyme slows down the rate of accumulation of NAP. Concomitantly or alternatively, it is also possible to upregulate the expression level of MAOB (monoamine oxidase), which shifts NAP to the downstream TCA cycle and provides energy for cell growth and protein production. Control strategy 3: Upregulate MAOB activity to shift NAP to the downstream TCA cycle and provide energy for cell growth and protein production. [Figure 11]Figure 1 shows the cell growth profile after upregulation of regulatory enzymes for the production of inhibitory metabolites. Control shows the cell culture control condition of a CHO K1 batch not transfected with any genes. T1 shows the cell growth profile when CHO K1 cell line was transfected with Got1. T2 shows the transfection of Got1 and Hoga1. T3 shows the transfection of Got1, Hoga1, Cat, and Slc35a1. After transfection, a dramatic increase in peak cell density was observed, with T2 and T3 showing a 50.9% increase in peak cell density compared to the control. [Figure 12] We present genetic engineering strategies targeting α-hydroxyisocaproic acid (HICA) accumulation and methylsuccinic acid (MSA) accumulation in CHO-K1 cells. In this study, metabolic by-products HICA and MSA, previously identified as accumulated growth inhibitors from batch and fed-batch CHO bioprocesses, are the targets of genetic engineering studies. Genome-scale modeling (GEM) reveals additional metabolic pathways that can be controlled by genetic engineering strategies. Inhibitor accumulation can be controlled by upregulating the expression of characterized genes (in italics) responsible for regulating the metabolic enzymes that catalyze the reactions. [Figure 13A]Figures 13A, 13B, 13C and 13D show genetic engineering strategies targeting the accumulation of trigonelline (TRI), N-acetylputrescine (NAP), trans-aconitic acid (TAA) and cytidine-5'-monophosphate (CMP) accumulation in the CHO-K1 bioprocess. Figure 13A shows the upregulation of Cat through the metabolism of tryptophan and the de novo synthesis of nicotinamide. Figure 13B shows the upregulation of Got1 and Hoga1 through the metabolism of proline. Figure 13C shows the upregulation of Got1 through the TCA cycle. Figure 13D shows the upregulation of Slc35a1 through the biosynthetic pathway of N-glycans. The accumulation of inhibitors can be controlled by upregulating the expression of characterized genes (in italics) responsible for regulating the metabolic enzymes catalyzing the reactions. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 14A] Figures 14A and 14B show the transfection efficiency demonstrated by GFP plasmid transfection. Here, the cellular physiology of CHO-S cells 2 days (Figure 14A) and 3 days (Figure 14B) after transfection was evaluated by flow cytometry assay. The results showed an increase in gene expression during peak production in the CHO-S cell line. [Figure 14B] Same as above. [Figure 15] Figure 15 shows CHO-S cells visualized under a fluorescent microscope. (i) Images of cells in normal lighting conditions. (ii) Images of cells visualized under fluorescent light. Shown in (i) and (ii): (A) cells transfected with GFP driving plasmid on day 2, (B) control cells without plasmid insertion on day 2, (C) cells transfected with GFP driving plasmid on day 3, (D) control cells without plasmid insertion on day 3. [Figure 16]Figures 16A, 16B, 16C, 16D, 16E, 16F, and 16G are graphs showing the expression levels of the seven different genes studied upon transfection in CHO-S cells. The study was divided into nine unique subclone groups. Each subclone was overexpressed with an individual gene of interest by transient transfection into the cells at a plasmid concentration of 50 μg μL-1. In a further group of gene mix subclones, all seven genes were transfected into the cells at once, but in each case the individual plasmid concentrations used were lower (6.25 μg μL-1 per gene, for a total plasmid concentration of 43.75 μg μL-1 for the seven genes). A negative control in which no plasmid was transfected was also included. Each subclone was cultured for six days, at which point the cells and cell culture supernatants were harvested. Cells from both control and transfected subclones were derived from the same culture to eliminate variability in growth cycles and other culture conditions. Gene expression levels were quantified by qPCR. [Figure 17-1]Figures 17A, 17B, 17C, 17D, 17E, 17F, and 17G are graphs showing the accumulation of inhibitory metabolites in the different subclones. The accumulated concentrations of metabolites are shown here: Figure 17A, CMP; Figure 17B, GMP; Figure 17C, HICA; Figure 17D, MSA; Figure 17E, NAP; Figure 17F, TAA; and Figure 17G, TRI. The study was divided into nine unique subclone groups. Each subclone was overexpressed with an individual gene of interest by transient transfection into cells at a plasmid concentration of 50 μg μL-1. In a further gene mix subclone group, all seven genes were transfected into cells at once, although the individual plasmid concentrations used in each case were lower (6.25 μg μL-1 per gene, for a total plasmid concentration of 43.75 μg μL-1 for the seven genes). A negative control in which no plasmid was transfected was also included. Each subclone was cultured for 6 days, at which point cells and cell culture supernatants were harvested. Cells from both control and transfected subclones were derived from the same culture to eliminate variability in growth cycles and other culture conditions. Quantification of inhibitory metabolite accumulation was performed by LC-MS. Bars, mean ± sem; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. Statistics by two-tailed t test. [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 18A]Figures 18A and 18B are graphs showing the growth profiles of the different subclones. Figure 18A shows the viable cell density (VCD) profile. Figure 18B shows the cumulative viable cell density (VCD) profile. In this study, each subclone was overexpressed with an individual gene of interest by transient transfection into cells at a plasmid concentration of 50 μg μL-1. In further gene mix subclones, all seven genes were transfected into cells, but in each case the individual plasmid concentrations used were lower (plasmid concentration of 6.25 μg μL-1 per gene). A negative control in which no plasmid transfection took place was also included in the study. Each subclone was cultured for 6 days and the cell culture supernatant was collected on the day of harvest. [Figure 18B] Same as above. [Figure 19A] Figures 19A and 19B are graphs showing the growth profiles of transfected subclones of multiple genes. Figure 19A shows the VCD profile. Figure 19B shows the integrated VCD profile. All seven key genes were transfected into host cells to test the overall function and their interactions. Gene mix subclones were overexpressed with all seven genes of interest by transient transfection at a plasmid concentration of 6.25 μg μL-1 per gene. A negative control in which no plasmid transfection occurred was also included in the study. Each subclone was cultured until cell viability dropped below 80%. Cells from both control and transfected subclones were derived from the same culture to eliminate variations in growth cycle and other culture conditions. [Figure 19B] Same as above. [Figure 20-1]Figures 20A, 20B, 20C, 20D, 20E, 20F, and 20G are graphs showing the nutrient uptake profiles of individual transfected subclones. Figure 20A, sodium; Figure 20B, potassium; Figure 20C, ammonia; Figure 20D, lactate; Figure 20E, glucose; Figure 20F, glutamate; and Figure 20G, glutamine profile. This study was divided into unique subclone groups. Each subclone was overexpressed with an individual gene of interest by transient transfection into cells at a plasmid concentration of 50 μg μL-1. A negative control in which no plasmid transfection occurred was also included in the study. Each subclone was cultured until cell viability dropped below 80%. Cells from both control and transfected subclones were derived from the same culture to eliminate variability in growth cycles and other culture conditions. A greater than 50% reduction in ammonia and less glucose consumption was observed in the engineered cell lines. [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 21-1] Figures 21A, 21B, 21C, 21D, 21E, 21F, and 21G are graphs showing nutrient uptake profiles of gene mix transfected subclones. Figure 21A, sodium; Figure 21B, potassium; Figure 21C, ammonia; Figure 21D, lactate; Figure 21E, glucose; Figure 21F, glutamate, and Figure 21G, glutamine profiles. All seven key genes were transfected into host cells to test global function and their interactions. Gene mix subclones were overexpressed with all seven genes of interest by transient transfection at a plasmid concentration of 6.25 μg μL-1 per gene. A negative control in which no plasmid transfection occurred was included. Each subclone was cultured until cell viability dropped below 80%. Cells from both control and transfected subclones were derived from the same culture to eliminate variations in growth cycle and other culture conditions. [Figure 21-2] Same as above. [Figure 21-3] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] These 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 primary workhorse for monoclonal antibody (mAb) production. To achieve high antibody productivity, manufacturing processes are performed under high cell density conditions (>10 million cells / mL). However, while cell densities are at record highs, productivity stagnates due to high concentrations of secreted inhibitory metabolites. The accumulation of these inhibitory metabolites impairs cell growth and also poses a risk to overall product quality, as the generation of waste by-products (e.g., ammonia) has been shown to alter key product properties of recombinant proteins. In this study, in addition to common inhibitors, novel metabolic biomarkers were identified and characterized by LC-MS metabolomics. The workflow for waste inhibitor metabolomics was: candidate identification, validation and quantification of inhibitory impact, and development of control strategies.

[0007] Novel biomarkers of protein production in CHO cells and other mammalian cells are aconitic acid (AA), more specifically aconitic acid (AA), leucic acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI) and N-acetylbutrecinium (NAP). The previously identified guanosine monophosphate (GMP) and indole-3-carboxylic acid (ICA) were also identified in this study and can be combined with the newly described biomarkers.

[0008] Described herein is a cell culture method in which the concentration in the cell culture medium of at least one metabolite selected from aconitic acid (AA), leucic acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI) and N-acetylputresinium (NAP) is maintained at a low level. In cell culture, particularly in high density cell cultures such as fed-batch and / or perfusion bioprocesses aimed at producing large quantities 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 effect of these metabolites can be limited by maintaining their concentration in the cell culture medium below a 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 embodiment, the cell culture method comprises (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 at or below an inhibitory concentration for the at least one metabolite in the cell culture medium. In one embodiment, 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 generate human therapeutic products such as HEK293 cells, HT-1080 cells, engineered T cells, and engineered natural killer cells.

[0011] The inhibitory amount of a metabolite may range from 1 nm to 50 mM, more specifically from 100 nM to 10 mM. When the metabolite is AA, the inhibitory concentration may 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 may be less than 10 μM, 23.5 μM, 47 μM, 100 μM, 1 mM or 3 mM. When the metabolite is a CMP, the inhibitory concentration may 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 may be less than 1 μM, 3.75 μM, 7.5 μM, 100 μM, 1 mM or 3 mM. When the metabolite is a TRI, the inhibitory concentration may 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 may be less than 0.1 μM, 0.3 μM, 0.6 μM, 100 μM, 1 mM or 3 mM. In one embodiment, the method comprises measuring the concentration of at least one metabolite. The concentration of the metabolite can be measured by any method known to the analytical profession.

[0015] Metabolite concentration can be measured once or multiple times during cell culture.In one embodiment, metabolite concentration is measured continuously, intermittently, every 30 minutes, every hour, every 2 hours, twice a day, every day, or every 2 days.In a preferred embodiment, metabolite concentration is measured every day.

[0016] The concentration may be measured by a method that is not automated and is not integrated into the cell culture method, for example, a measurement method that involves manually taking samples from the cell culture medium and measuring the specific concentration in the sample may be used. Alternatively, determining the concentration of metabolites may be automated and integrated into the cell culture method.

[0017] Exemplary methods for measuring the concentrations of metabolites include 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 combinations thereof. For example, in GC-MS, experiments can be performed using an autosampler that takes 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 more combinations of the aforementioned methods.

[0018] In one embodiment, when the measured concentration of one or more metabolites exceeds a predetermined value, such as an inhibitory concentration, a control strategy is used to limit the concentration of the inhibitory metabolite. Control strategy 1 reduces the concentration of at least one metabolite precursor (e.g., amino acid, glucose) in the cell culture medium 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 so 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 precursor is 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 aspartate, tryptophan, and / or glutamine. When the metabolite is NAP, the precursors are arginine, proline, aspartate, glutamine, and / or asparagine.

[0022] Combinations of the aforementioned precursors may also be reduced in the process. Reducing the concentration of the inhibitory metabolite precursor allows for the reduction of the inhibitor concentration to optimize the process.

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

[0024] The concentration of the precursor may range from 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 optimal precursor concentrations from the above concentration ranges to minimize inhibitory metabolite accumulation.

[0030] Reducing the amount of glucose can include culturing cells at low glucose concentrations, 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 the pyruvate carboxylase enzyme, or using inhibitors (small molecule or protein based) against signaling pathways (AKT, mTOR, HIF1a, etc.) that regulate the activity of energy metabolic pathways (glycolysis, TCA cycle, redox pathways).

[0031] In control strategies 2a and b and 3, upstream and downstream enzyme expression (strategy 2) or activity (strategy 3) are manipulated to alleviate the synthesis of inhibitory metabolites. The general idea behind control strategies 2 and 3 is to downregulate upstream enzymes to make cells produce less inhibitory metabolites and / or upregulate downstream enzymes to make cells consume metabolites to generate energy. Figure 10 shows three control strategies illustrated for NAP, Figure 12 shows control strategies for MSA and HICA, Figure 13A shows control strategies for TRI, Figure 13B shows further control strategies for NAP, Figure 13C shows control strategies for TAA, and Figure 13D shows control strategies for CMP.

[0032] In control strategy 2a, upstream and / or downstream enzyme expression that controls the level of a metabolite can be modulated using inhibitory nucleic acids such as siRNA. The term "inhibitory nucleic acid" refers to RNA, such as single-stranded or double-stranded RNA or DNA, specifically triple-stranded oligonucleotides, ribozymes, aptamers, small interfering RNA, including siRNA (short interfering RNA) and shRNA (short hairpin RNA), antisense RNA or portions thereof, or analogs or mimetics thereof, that can reduce or inhibit the expression of a target gene or sequence. Inhibitory nucleic acids can act, for example, by mediating degradation or by inhibiting translation of mRNA complementary to the interfering RNA sequence. Administration of the inhibitory nucleic acid to a mammalian cell reduces (e.g., 5%, 10%, 25%, 50%, 75%, or 90-100%) the expression (e.g., transcription or translation) of the target sequence. Typically, a nucleic acid inhibitor comprises or corresponds to at least a portion of a target nucleic acid molecule or its ortholog, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. The inhibitory nucleic acid may have substantial or complete identity to a target gene or sequence, or may comprise a mismatch region (i.e., a mismatch motif). The sequence of the inhibitory nucleic acid may correspond to a full-length target gene or a subsequence thereof. In one embodiment, 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 level of the metabolite, resulting in reduced levels of the metabolite. In control strategy 3, enzyme activity can be controlled by two different approaches.

[0034] Approach 1: Regulating enzyme expression to reduce metabolite synthesis involves adding an inhibitor of enzyme activity. Exemplary inhibitors of enzyme activity include small molecules, peptides, proteins, and nucleic acids that bind to the active site of the enzyme and block the activity of the enzyme.

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

[0036] Regarding 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 ADH5, BCAT1, SLC35A1, 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, CAT, ADH5, BCAT1, GOT1, HADHA / B, or a combination thereof; the metabolite is MSA and the enzyme is BCAT1, GOT1, ETHE1, AMT, HADHA / B, MMUT, HOGA1, SLC35A1, or a combination thereof; the metabolite is TRI and the enzyme is BCAT1, GOT1, HADHA / B, NADSYN1, NNMT, CAT, HOGA1, SLC35A1, NMNAT1, SULT4A1, or a combination thereof; the metabolite is NAP and the enzyme is BCAT1, SLC35A1, CAT, HADHA / B, SAT1 / 2, HOGA1, AMD1, ODC1, GOT1, MAOB, or a combination thereof; the metabolite is a TAA and the enzyme is BCAT1, ADH5, GOT1, HOGA1, SLC35A1, HADHB, or a combination thereof; or It's a combination of those.

[0037] In one embodiment, the enzyme comprises ADH5, BCAT1, CAT, GOT1, HADHB, HOGA1, SLC35A1, or a combination thereof. the metabolite is HICA and the enzyme comprises ADH5, BCAT1, SLC35A1, GOT1, HADHB, or a combination thereof; the metabolite is CMP and the enzyme comprises ADH5, BCAT1, CAT, GOT1, HOGA1, SLC35A1, HADHB, or a combination thereof; the metabolite is MSA and the enzyme comprises BCAT1, GOT1, HADHB, HOGA1, SLC35A1, or a combination thereof, or comprises a combination thereof; the metabolite is TRI and the enzyme comprises BCAT1, GOT1, HADHB, CAT, HOGA1, SLC35A1, or a combination thereof; the metabolite is NAP and the enzyme comprises BCAT1, SLC35A1, HOGA1, GOT1, CAT, HADHB, or a combination thereof; the metabolite is a TAA and the enzyme is BCAT1, ADH5, GOT1, HOGA1, SLC35A1, HADHB, or a combination thereof; or A combination of them.

[0038] In some embodiments, the enzymes include ADH5, BCAT1, GOT1, HADHA / B, HOGA1, SLC35A1, and CAT. In control strategy 4, for at least one metabolite, maintaining the at least one metabolite in the cell culture medium below an inhibitory concentration includes controlling process parameters including temperature, dissolved oxygen level, pH, or a combination thereof. The optimum temperature, dissolved oxygen level, and pH can be determined by the rate of enzyme reaction and inhibitory metabolite accumulation. The process can be optimized for fed-batch and perfusion bioprocesses.

[0039] Exemplary temperatures are from about 30°C to about 40°C, specifically temperatures of 31°C, 32°C, 33°C, 34°C, 35°C, 36°C and 37°C during the early and late phases of cell culture.

[0040] Exemplary pH ranges from pH 5.5 to 8.5, e.g., pH 6.5, 6.7, 6.9, 7, 7.2, 7.4, or 7.6. CO2 sparging and / or Na2CO3 supplementation can be used to balance the pH. Exemplary CO2 sparging rates include 0.3 SLPH, 0.4 SLPH, 0.5 SLPH, 0.6 SLPH, and 0.7 SLPH (standard liter per hour). Exemplary agitation rates for Na2CO3 supplementation include 90 RPM, 120 RPM, 150 RPM, and 170 RPM (revolutions per minute).

[0041] Exemplary dissolved oxygen set points include 5% to 50%, for example, 20%, 30% and 40%. Any cell that can be grown in cell culture can be used in the methods described herein, hi some embodiments, the cell is a mammalian cell. Non-limiting examples of mammalian cells include BALB / c mouse myeloma line (NSO / I, ECACC number: 85110503); human retinoblast cells (PER.C6, CruCell, Leiden, The Netherlands); SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney lines (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-1 587); human cervical cancer cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC5 cells; FS4 cells; and human hepatocellular carcinoma line (HepG2). In one embodiment, the cells are CHO cells, HEK293 cells, HT-1080 cells, genetically engineered T cells, and genetically engineered natural killer cells. Genetically engineered T cells include CAR T cells.

[0042] Any number of commercially and non-commercially available hybridoma cell lines can be utilized in the methods described herein. The term "hybridoma" as used herein refers to a cell or cell progeny resulting from the fusion of an immortalized cell with an antibody-producing cell. Such resulting hybridomas are immortalized cells that produce antibodies. The individual cells used to make hybridomas can be derived from any mammalian source, including but not limited to rats, pigs, rabbits, sheep, goats, and humans. In some embodiments, hybridomas are trioma cell lines that arise when the progeny of a heterohybrid myeloma fusion, which is the product of the fusion of a human cell with a mouse myeloma cell line, is subsequently fused with a plasma cell. In some embodiments, hybridomas are any immortalized hybrid cell line that produces antibodies, such as, for example, quadromas. One skilled in the art will understand that hybridoma cell lines may have different nutritional requirements and / or may require different culture conditions for optimal growth, and may modify the conditions as necessary.

[0043] As used herein, the terms "culture" and "cell culture" refer to a cell population suspended in a medium under conditions suitable for the survival and / or growth of the cell population. As will be apparent to one of skill in the art, in some embodiments, these terms as used herein refer to a combination comprising a cell population and a medium in which the population is suspended. In some embodiments, the cells of the cell culture comprise mammalian cells.

[0044] The methods described herein can be used with any cell culture method suitable for the desired process (e.g., production of a recombinant protein (e.g., an antibody)). As a non-limiting example, 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 the expressed protein (e.g., an antibody) is then harvested. Alternatively, as another non-limiting example, cells may be grown in batch-refeed, where the culture is not terminated but rather new 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 practice the methods described herein.

[0045] 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 at one or more times after the start of the culture process. Such provided components typically include nutritional components of the cells that are depleted during the culture process. Fed-batch cultures are typically stopped at some point, and the cells and / or components in the medium are harvested and optionally purified. In some embodiments, fed-batch cultures include a basal medium supplemented with a feed medium.

[0046] Alternatively, cells can be grown in a perfusion process, in which cells are maintained in a bioreactor with continuous removal of cellular waste and depleted medium. The rate at which new medium is added is approximately the same as the rate at which depleted medium is removed from the system. Removal of spent medium can be accomplished by alternating tangential flow, standard tangential flow filtration, hollow fiber filtration, etc.

[0047] The cells may be grown in any convenient volume selected by the practitioner. For example, the cells may be grown in small scale reaction vessels ranging in volume from a few milliliters to a few liters. Alternatively, the cells may be grown in large scale commercial bioreactors ranging in volume from 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 therebetween.

[0048] The temperature of the cell culture is selected primarily based on the range of temperatures at which the cell culture remains viable and at which high levels of the desired product (e.g., recombinant protein) are produced. In general, most mammalian cells can grow well and produce the desired product (e.g., recombinant protein) within a range of about 25°C to 42°C, although the methods described herein are not limited to these temperatures. Certain mammalian cells can grow well and produce the desired product (e.g., recombinant protein or antibody) within a range of about 35°C to 40°C. In certain embodiments, the cell culture is grown at a temperature of 20-45°C at one or more times during the cell culture process. One of skill in the art will be able to select the appropriate temperature(s) at which to grow the cells depending on the particular needs of the cells and the particular production requirements of the practitioner. The cells may be grown for any length 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.

[0049] In some embodiments, cells are grown for longer or shorter times 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, cells are grown for a sufficient time to achieve a predetermined cell density. In some embodiments, cells are grown for a sufficient time to achieve a cell density that is a predetermined percentage of the maximum cell density that the cells would eventually reach if grown undisturbed. For example, cells can be grown for a sufficient period of time to achieve a desired viable cell density of 1-99% of the maximum cell density. In some embodiments, 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, cells are grown until the cell density does not increase by more than 5% per day of culture.

[0050] In some embodiments, the cells are allowed to grow for a defined period of time. For example, depending on the starting concentration of the cell culture, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, the cells may be grown for 0-20 days or more, specifically 4-10 days. In some cases, the cells may be grown for a month or more. The practitioner can select the duration of the initial growth phase depending on the requirements of protein production and the needs of the cells themselves.

[0051] The cell culture may be agitated or shaken during the initial cultivation phase to increase oxygenation and distribution of nutrients to the cells. One skilled in the art will appreciate 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.

[0052] 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, so that a metabolic shift occurs in the culture. The metabolic shift can be achieved, for example, by changing the temperature, dissolved oxygen level, pH, osmolality, 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 known in the art, temperature shift is not the only mechanism that can achieve a suitable metabolic shift. For example, such metabolic shift can also be achieved by shifting other culture conditions, including but not limited to pH, dissolved oxygen, osmolality, and sodium butyrate level. 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.

[0053] When shifting the temperature of a culture, the temperature shift can be relatively gradual. For example, the temperature shift can take several hours or days to complete. Alternatively, the temperature shift can be relatively rapid. For example, the temperature shift can be completed within a few hours. Given appropriate production and control equipment, such as is standard in large-scale commercial production of polypeptides or proteins, the temperature shift can be completed in less than an hour.

[0054] In some embodiments, once the cell culture conditions have been shifted as described above, the cell culture is maintained for a subsequent production phase under a second set of culture conditions that are conducive to the survival and viability of the cell culture and suitable for expression of the desired polypeptide or protein at commercially relevant levels.

[0055] As mentioned 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, osmolality, and sodium butyrate levels. 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 mentioned above, multiple individual temperature shifts can be used to increase cell density or viability or to increase recombinant protein expression.

[0056] The cells can express a recombinant protein, a gene product, or a 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.

[0057] The methods described herein can be used to improve cell growth at high cell densities in high density cell cultures. As used herein, high cell density refers to cell growth at cell densities of 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 x 10 8 Cell density greater than 1 x 10 cells / mL, preferably 1 x 10 7 More than 5 x 10 cells / mL, more preferably 7 Refers to cell density in excess of cells / mL.

[0058] In some embodiments, cell growth is determined by viable cell density (VCD), maximum viable cell density, or integrated viable cell count (IVCC, also referred to as integrated viable cell density). In some embodiments, cell growth is determined by maximum viable cell density. The term "viable cell density" as used herein refers to the number of cells present in a given volume of 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®. The term maximum cell density as used herein refers to the maximum cell density achieved during cell culture. The term "cell viability" as used herein 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) can be used that does not pass through the membrane of living cells but can pass through the disrupted membrane of dead or dying cells.

[0059] As used herein, the term "Integrated viable cell count (IVCC)" or "Integrated viable cell density (IVCD)" 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 instants t and t+1. t=0 can be considered negligible. t and VCDs t+1 is the viable cell density at time points t and t+1.

[0060] As used herein, the term "titer" refers to the total amount of recombinantly expressed protein produced by, for example, a cell culture in a given amount of medium volume. Titer is typically expressed in grams of protein per liter of medium.

[0061] In some embodiments, cell growth is increased by at least 5%, 10%, 15%, 20% or 25% compared to the control culture. The control culture may be identical to the culture described above, except that it is not cultured with step (ii). In some embodiments, cell growth is increased by at least 10% compared to the control culture. In some embodiments, cell growth is increased by at least 20% compared to the control culture.

[0062] In some embodiments, productivity is determined by titer and / or volumetric productivity. The term "titer" as used herein refers to, for example, the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titer is typically expressed in grams of protein per liter of medium. In some embodiments, productivity is determined by titer. In some embodiments, productivity is increased by at least 5%, 10%, 15%, 20% or 25% compared to a control culture. In some embodiments, productivity is increased by at least 10% compared to a control culture. In some embodiments, productivity is increased by at least 20% compared to a control culture.

[0063] 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 x 10 8 In some embodiments, the maximum cell density of the cell culture is greater than 5×10 6 In some embodiments, the maximum cell density of the cell culture is greater than 1×108 Greater than cells / mL.

[0064] The terms "medium", "cell culture medium" and "culture medium" as used herein refer to a solution containing nutrients that nourish growing mammalian cells. Typically, such solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and / or survival. Such solutions may also contain supplemental components that enhance growth and / or survival above a minimal rate, including but not limited to hormones and / or other growth factors, specific ions (sodium, chloride, calcium, magnesium, phosphate, etc.), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds that are usually present at very low final concentrations), inorganic compounds (e.g., iron) that are present at high final concentrations, amino acids, lipids, and / or glucose or other energy sources. In some embodiments, the medium is advantageously formulated at a pH and salt concentration that is optimal for cell survival and proliferation. In some embodiments, the medium is a feed medium that is added after the initiation of cell culture.

[0065] A wide variety of mammalian growth media can be used according to the methods described herein.In some embodiments, 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, cells can be grown in complex medium, in which not all components of the medium are known and / or controlled.

[0066] Chemically defined growth media for mammalian cell culture have been extensively developed and published over the past several decades. All components of a defined medium are well characterized, and therefore, a defined medium does not contain complex additives such as serum or hydrolysates. Early media formulations were developed to allow cell growth and maintenance of viability with little or no concern for protein production. More recently, media formulations have been developed with the express purpose of supporting highly productive recombinant protein producing cell cultures. Such media generally contain high amounts of nutrients, especially amino acids, to support cell growth and / or maintenance at high density. If necessary, these media may be modified by one of skill in the art for use in the methods described herein.

[0067] Because not all components of a complex medium are fully characterized, a complex medium may contain, inter alia, simple and / or complex carbon sources, simple and / or complex nitrogen sources, and additives such as serum. In some embodiments, a complex medium contains additives such as hydrolysates in addition to other components of a defined medium as described herein.

[0068] 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, but others do not require protein components and are therefore called protein-free media. Typical chemical components of media fall into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and other categories that are not neatly classified.

[0069] Supplementary components may be added to the cell culture medium optionally. The term "supplementary components" as used herein refers to components that enhance growth and / or survival above a minimum 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 that are usually present at very low final concentrations), amino acids, lipids, and / or glucose or other energy sources. In some embodiments, the supplementary components may be added to the initial cell culture. In some embodiments, the supplementary components may be added after the initiation of cell culture.

[0070] Typically, trace elements refer to various inorganic salts that are included at submicromolar levels. 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 initial cell culture medium at micromolar concentrations. Manganese is also often included in trace elements as a divalent cation (MnCl2 or MnSO4) in the nanomolar to micromolar range. Many less common trace elements are usually added at nanomolar concentrations.

[0071] In some embodiments, the medium used in the method supports high cell densities in cell culture, e.g., 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 x 10 8 The medium is suitable for supporting cells / mL. In some embodiments, the cell culture is a mammalian cell fed-batch culture, preferably a CHO cell fed-batch culture.

[0072] In many cases, cells are selected or engineered to produce high levels of a desired product (e.g., a recombinant protein or antibody). In many cases, cells are engineered to produce high levels of recombinant protein, for example, by introduction of a gene encoding the protein of interest and / or introduction of genetic control elements (whether endogenous or introduced) that regulate expression of that gene.

[0073] Certain proteins may have detrimental effects on cell growth, cell viability, or other characteristics of cells that ultimately limit the production of the protein of interest in some way. Even among a population of one particular type of cells 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 proteins with higher activity levels (e.g., enzyme activity). In certain embodiments, cell lines are empirically selected by the practitioner to ensure that they grow 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 expressed protein, or any combination of these, or any other conditions deemed important by the practitioner.

[0074] 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 that is engineered to produce a protein of interest as described herein. The protein can be expressed from a gene that is endogenous to the cell or from a heterologous gene that is introduced into the cell. The protein can be a naturally occurring protein or can have a genetically engineered or selected sequence.

[0075] Proteins that may be desired to be expressed are often selected based on interesting or useful biological or chemical activity. For example, the method can be used to express any pharma- ceutically or commercially relevant enzymes, receptors, antibodies, hormones, regulators, antigens, binders, etc. In some embodiments, the protein expressed by the cells in culture is selected from an antibody or fragment thereof, a nanobody, a single domain antibody, a glycoprotein, a therapeutic protein, a growth factor, a clotting factor, a cytokine, a fusion protein, an active pharmaceutical ingredient, a vaccine, an enzyme, or Small Modular ImmunoPharmaceuticals™ (SMIPs). Those skilled in the art will understand that any protein that can be expressed can select the particular protein to be produced based on the particular needs of the skilled artisan.

[0076] According to the method of the present invention, the production of antibodies is of particular interest. Antibodies are proteins that have 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 expressed antibody is a monoclonal antibody.

[0077] In some embodiments, the monoclonal antibody is a chimeric antibody. A chimeric antibody contains amino acid fragments derived from multiple organisms. A chimeric antibody molecule can contain, for example, an antigen-binding domain from a mouse, rat, or other species antibody with a human constant region.

[0078] In some embodiments, the monoclonal antibodies are human antibodies derived, for example, through the use of ribosome display or phage display libraries, or the use of xenografts in which the native antibody genes have been inactivated and functionally replaced with human antibody genes, while leaving other components of the natural immune system intact.

[0079] 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, thus minimizing any potential immune reaction when delivered to a human subject. In a humanized antibody, the amino acid residues in the complementarity determining region are at least partially replaced with residues from non-human species that provide the desired antigen specificity or affinity. Such modified immunoglobulin molecules can be produced by any of several techniques known in the art.

[0080] In some embodiments, the monoclonal, chimeric, or humanized antibody may contain amino acid residues that do not naturally occur in any antibody in any natural species. These foreign residues can be utilized, for example, to provide the monoclonal, chimeric, or humanized antibody with new or modified specificity, affinity, or effector function. In some embodiments, the antibody may be conjugated to drugs for systemic drug therapy, such as toxins, low molecular weight cytotoxic drugs, biological response modifiers, and radionuclides.

[0081] Generally, the nucleic acid molecule introduced into the cell encodes a protein that is desired to be expressed. Alternatively, the nucleic acid molecule may encode a gene product that induces the cell to express the desired protein. For example, the introduced genetic material may encode a transcription factor that activates the transcription of an endogenous or heterologous protein. Alternatively, or additionally, the introduced nucleic acid molecule may increase the translation or stability of a protein expressed by the cell. Suitable methods for introducing sufficient nucleic acid into a mammalian host cell to achieve expression of a protein of interest are known in the art. For mammalian cells, common methods for introducing genetic material into mammalian cells include the calcium phosphate precipitation method or the Lipofectamine™ method.

[0082] In some embodiments, the nucleic acid introduced into the cell is in the form of a naked nucleic acid molecule. For example, the nucleic acid molecule introduced into the cell may consist of only the nucleic acid encoding the protein and the necessary genetic control elements. Alternatively, the nucleic acid encoding the 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 the cell is contained within a viral vector. For example, the nucleic acid encoding the protein can be inserted into the viral genome (or a partial viral genome). The regulatory elements directing the expression of the protein 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.

[0083] 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 an appropriate buffer and subjecting the cells to a high voltage electric pulse (e.g., by electroporation). A further method for introducing naked DNA into cells is by mixing the DNA with a liposome suspension containing cationic lipids. 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 to a cation such as polylysine bound to a ligand of a cell surface receptor. Binding of the DNA-ligand complex to the receptor promotes uptake of the DNA by receptor-mediated endocytosis.

[0084] Defective retroviruses have been well characterized for use in gene transfer for gene therapy purposes. A nucleic acid encoding a protein of interest can be inserted into the retroviral genome to construct a recombinant retrovirus. In addition, a portion of the retroviral genome can be removed to render the retrovirus replication defective. Such replication-defective retroviruses are then packaged into virions that can be used to infect target cells through the use of a helper virus by standard techniques. For example, the genome of an adenovirus can be engineered to encode and express a protein of interest, but inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Exemplary adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Adeno-associated virus (AAV) is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and productive life cycle.

[0085] If the method used to introduce nucleic acid molecules into a cell population results in the modification of a large proportion of cells and efficient expression of 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 protein by the cell population so that the population can be immediately used to seed 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 produces protein.

[0086] 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 processing 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 a 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. The expression of such a transcription factor in turn leads to the expression, or stronger expression, of the protein of interest.

[0087] In general, it will typically be desirable to isolate and / or purify the recombinant proteins, gene products, or cell products 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, for example, by centrifugation or filtration, as a first step in the purification process.

[0088] Alternatively, the expressed protein may be bound to the surface of the host cell. For example, the medium may be removed and the host cells expressing the protein may be lysed as a first step in the purification process. Lysis of mammalian host cells may be accomplished by any number of means well known to those skilled in the art, including physical disruption with glass beads and exposure to high pH conditions.

[0089] The expressed proteins 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 by any other available technique for purification of proteins.

[0090] In certain embodiments, the produced polypeptide or protein will have pharmacological activity and 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 pharma- ceutically acceptable carrier. As used herein, the term "pharma-ceutically 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.

[0091] The invention is further illustrated by the following non-limiting examples. EXAMPLES

[0092] Example 1: Identification of inhibitors The entire CHO metabolome was screened using a HILIC liquid chromatography column Orbitrap™ mass spectrometry (LC-MS). 30,000 features were analyzed and over 1000 features were accumulated throughout the cell culture. The inhibitory effect of the top 20 features was tested analytically and biologically. The metabolites structurally confirmed by LC-MS are shown in Table 1. Concentrations at the end of the culture were also measured using LC-MS with different methods.

[0093] Table 1: Structurally confirmed metabolites at the culture supernatant concentrations at the end of the culture.

[0094] [Table 1]

[0095] Example 2: Standard bioprocessing and biological confirmation of metabolites CHO K-1 industry standard batch process: NIH CHO-K1 cell line was cultured for 6 days and 6 mM glutamine supplement was added on the day of inoculation. The working volume is 30 mL in a 125 mL shake flask, and the inoculation cell density is 500,000 cells / mL. The shaking incubator parameters are 125 RPM and 5% CO2.

[0096] CHO GS Industry Standard Batch Process: CHOZn® cell line is cultured for 6 days. Working volume is 30 mL in 125 mL shake flasks with inoculation cell density of 500,000 cells / mL. Shaking incubator parameters are 125 RPM and 5% CO2.

[0097] HEK293 industry standard batch process: Cultivate MBL HEK293 cell line for 6 days. Working volume is 30 mL in 125 mL shake flasks, inoculation cell density is 500,000 cells / mL, 6 mM glutamine supplement was added on the day of inoculation. Shaking incubator parameters are 125 RPM and 5% CO2.

[0098] Cells were exposed to the inhibitory metabolites identified in Table 1 at their terminal concentrations in batch culture for 6 days. AA, ICA, CMP and mixture conditions showed lower cell density than the control group (Figure 3). All inhibitory metabolites reduced antibody productivity (Figure 4).

[0099] Industrial standard fed-batch process: CHO-K1 cell line is cultured in the corresponding medium. On the day of inoculation, 6 mM glutamine supplement is added and cultured for 14 days (feed). Working volume is 30 mL in 125 mL shake flasks, inoculation cell density is 500,000 cells / mL. Shaking incubator parameters are 125 RPM and 5% CO2. Feeding strategy was to supply 10% of the corresponding feed every day from day 3 to day 13.

[0100] In a standard industrial fed-batch process, cells were exposed to inhibitors for extended periods of time. This experiment mimics a therapeutic antibody production process, spiking the inhibitor at the cell concentration at the end of the culture. ICA was confirmed as a known inhibitor and was used as a positive inhibitor control. The mixed conditions excluded ICA and included AA, HICA, CMP, MSA, TRI, and NAP. The process was continued for 14 days, and from day 6, cell density under inhibitor conditions was lower than the negative control batch. The results are shown in Figure 3. Compared to the control batch, CMP, ICA, TRI, and HICA showed a stronger growth inhibitory effect, resulting in a 40-50% VCD reduction with peak VCD on day 9.

[0101] Example 3 shows the impact on metabolite productivity and quality. IgG titers were measured by HPLC using a Protein A column. Product quality was quantified by the IgG glycan profile measured by HPLC using a Glycan Column.

[0102] All seven metabolites showed inhibitory effects on productivity and product quality. HICA, CMP, ICA, and TRI showed strong inhibition on productivity, with more than 30% productivity loss compared to the control (Figure 4). Product quality is an important parameter for bioprocessing and drug approval, and glycan profile is one of the most important critical quality attributes (CQAs). AA, HICA, CMP, MSA, and ICA showed higher effects on G1F formation (Figure 5), AA completely inhibited G2F formation, and all other six inhibitors showed significant effects on G2F formation (Figure 6). Replicate spiking experiments showed that the effects on glycan profile were consistent (Figures 7, 8, and 9).

[0103] conclusion More than 30,000 features were captured by the LC-MS metabolomics study described herein, and the top 20 inhibitor candidates with high confidence in identification matching were selected by multivariate data analysis and pathway analysis. The top candidates were experimentally validated by spiking into batch cultures, and 11 of 15 of them showed inhibitory effects on cell growth. Eight metabolic by-products were structurally and biologically validated. 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).

[0104] Table 2: AA, HICA, CMP, GMP, MSA, ICA, TRI and NAP were identified as inhibitors of CHO cell processes in this study. GMP and ICA have been reported as CHO cell culture inhibitors in publications / patents, and the remaining six metabolites are claimed in this application. TRI, NAP, AA and MSA were identified for the first time as CHO cell metabolites.

[0105] [Table 2]

[0106] Example 4: Accumulation of inhibitory metabolites in fed-batch cultures The fed-batch conditions were the same as the CHO K-1 industry standard fed-batch culture process of Example 2. The batch process conditions were the same as the CHO K-1 industry standard batch process of Example 2.

[0107] Metabolites were identified by LC-ESI-MS. Table 3 shows the identified inhibitory metabolites that accumulated in the process and their accumulation after 6 days in the batch process and after 5, 8 and 14 days in the fed-batch process.

[0108] Table 3: Accumulation of inhibitory metabolites in batch and fed-batch processes

[0109] [Table 3]

[0110] This example shows inhibitor concentrations in batch and fed-batch cultures. The inventors concluded that inhibitory metabolites accumulated in the primary bioprocess. Example 5: Accumulation of inhibitory metabolites in CHO K1, CHO GS and HEK293 in batch cultures The batch process conditions were the same as in Example 2 for CHO K-1, CHO GS and HEK293 industrial standard batch processes. The 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 the batch cultures.

[0111] Table 4: Accumulation of inhibitory metabolites in CHOK1, CHOGS, and HEK293 in batch cultures.

[0112] [Table 4]

[0113] From the final accumulation of batch cultures, we conclude that inhibitory metabolites are widely present in mammalian cell lines. Example 6: Process Development to Mitigate Accumulation of Inhibitory Metabolites Transfection of inhibitory metabolite regulator genes into batch cultured CHO K1 cell line alleviates the growth of inhibitory metabolites.

[0114] The purpose of transfection is to overexpress the inhibitory metabolite regulatory genes / enzymes. The downstream enzyme-encoding genes were introduced into the CHO cell line by plasmids constructed with Got1, Hoga1, Cat, and Slc35a1 in pcDNA3.1(+) vector (V79020, ThermoFisher Scientific). The plasmids were amplified using 5α-competent E. coli (C2987I, New England BioLabs). The plasmids were transfected into mammalian cell lines using polyethylenimine (408727, Millipore Sigma) to alleviate the inhibitory metabolite concentration by overexpressing the inhibitory metabolite regulatory genes. The transfection was the same as the CHO K1 industry standard batch process, except that the initial inoculation cell density was 1 million cells / mL.

[0115] In Table 5 and Figure 11, control indicates the CHO K1 batch cell culture control condition without any gene transfection; T1 indicates the cell growth profile of CHO K1 cell line transfected with Got1; T2 indicates Got1 and Hoga1 transfection; and T3 indicates Got1, Hoga1 and Cat, Slc35a1 transfection. As shown in Table 5 and Figure 11 compared to the control cell culture, overexpression of inhibitory metabolite regulating genes in CHO K1 cell line reduces the concentration of all inhibitory metabolites and improves the growth profile.

[0116] Table 5: Transfection of inhibitory metabolite-regulating genes into CHOK1 cell line in batch culture

[0117] [Table 5]

[0118] From this data, it was concluded that inhibitor concentrations were reduced following transfection of inhibitory metabolite regulating genes into the CHO cell line. Example 7. Method development by pathway analysis combined with genome-scale modeling Screening of target genes was performed by batch mode cultivation of CHO cells under standard settings and operating conditions (no perturbation). Accumulation / depletion concentrations in the extracellular environment of major nutrients, including essential amino acids and glucose, were continuously measured by LC-MS during the cultivation period, and based on the results, the respective metabolic uptake fluxes were calculated and used as constraint inputs for GEM of the CHO-K1 cell line. A flux balance analysis was then performed with an objective function maximizing biomass to establish a baseline evaluation of growth and biomass productivity. Subsequently, each gene encoded in the gene library included in the model was upregulated, allowing a re-evaluation of the objective function at the end of each successful iteration. As a result of the study, several genes showing increased productivity relative to biomass were observed, suggesting that internal fluxes were efficiently reallocated to energy production for growth while reducing the generation of waste and metabolic by-products. Finally, by combining the computer-aided simulation work with the identification results obtained in reported publications where by-product metabolites were biologically confirmed to inhibit growth in CHO cultures, genes characterized as both contributing to the bioprocess by promoting growth in terms of biomass when compared to the established baseline, and also involved in biochemical processes consuming the identified metabolites, were further considered for genetic engineering validation studies.

[0119] material and method Plasmid construction for validation of inhibitory effect control. Biological validation of the control strategy towards metabolite accumulation was performed by transient transfection. The overall detailed strategy for subclone development and gene functionality analysis is shown below. Forward and reverse cloning primers were designed for each corresponding GOI. To ensure the expression of the enzyme, the pCMV promoter was incorporated into the plasmid to drive transcription. Furthermore, a Kozak sequence was added to drive translation activity. All the plasmids with restriction enzymes and primers incorporated in this study are shown in Table 6.

[0120] Table 6: Detailed workflow for subclone development and gene functionality analysis. Genes of interest (GOI) were first cloned from the CHO DNA genome, and then each GOI was subjected to the following steps of genetic manipulation studies for gene functionality analysis in CHO cells.

[0121] [Table 6]

[0122] Construction of plasmid vectors. CHO-K1 cells were extracted on days 4-6 of fed-batch culture. CHO-K1 RNA isolation and purification was performed using the RNeasy Mini Kit from Qiagen USA (Germantown, MD). Quantification of RNA concentration was performed with a NanoDrop™ OneC from Thermo Fischer Scientific (Waltham, MA). Construction of the resulting CHO-K1 cDNA library was performed utilizing the SS3 superscript kit from Invitrogen (Carlsbad, CA). PCR amplification was performed on the previously extracted cDNA. GOIs include Adh5, BCat1, Cat, Got1, Hadhb, Hoga1, Slc35a1. Primers were designed based on gene sequences and plasmid restriction enzyme sites obtained from the NCBI database (Appendix A). Detailed information on primer sequences is shown in Table 7. To amplify the GOI, PCR was performed using Q5® High-Fidelity DNA polymerase from New England Biolabs (Ipswich, MA). The PCR products were purified using DNA gel electrophoresis at 100V for 80 min and the QIAquick® Gel Extraction Kit from Qiagen USA (Germantown, MD). The complex GOI / plasmid was constructed using the GOI and pcDNA3.1 / Zeo(+) plasmid from Invitrogen (Carlsbad, CA). The complex GOI / plasmid was further digested with restriction enzymes from New England Biolabs (Ipswich, MA). After dephosphorylation of the 5'-ends of the plasmids using Quick CIP from New England Biolabs (Ipswich, MA), the complex GOI / plasmid was ligated using T4 DNA ligase from New England Biolabs (Ipswich, MA).

[0123] Table 7: Primer sequences designed for PCR amplification. In this study, seven genes were studied for enzymatic activity upon upregulation due to transient transfection, and the specific primer sequences for the forward and reverse primers for each are shown.

[0124] [Table 7]

[0125] Plasmid transformation. Bacterial transformation was performed using 50 μl of NEB® 5-alpha competent E. coli cells from New England Biolabs (Ipswich, MA) with 5 μL of previously ligated plasmid. The cells were carefully mixed by tapping five times on a mixing tube and incubated on ice for 30 minutes. The cells were heat shocked at 42°C for 30 seconds using a dry bath from Fisher Scientific (Waltham, MA) and the cells were placed back on ice and incubated for 5 minutes. A total volume of 950 μL of SOC Outgrowth medium from New England Biolabs (Ipswich, MA) was added to the cells and the total mixture was transferred to a 50 mL centrifuge tube from VWR (Radnor, PA). The tube was loosely capped to allow cellular respiration and incubated for 90 minutes in a humidified shaking incubator from Queue Systems (Columbia, SC) operated at 250 RPM and 37°C. A total volume of 100 μL of cells was evenly and gently spread using a disposable inoculation loop from VWR (Radnor, PA) onto LB agar ampicillin plates from Sigma Aldrich (St. Louis, MO) pre-warmed to 37° C. Cells were incubated overnight at 37° C.

[0126] Colonies (approximately 3–4) were selected after the incubation period and mixed with 5 mL of LB broth medium (Sigma Aldrich, St. Louis, MO) and 5 μL of ampicillin in a 50 mL centrifuge tube (VWR, Radnor, PA). The tubes were loosely capped to allow cellular respiration and incubated for 12 h in a humidified shaking incubator (Queue Systems, Inc., Columbia, SC) operated at 250 RPM and 32 °C. After 12 h of incubation, plasmid extraction was performed using the ZymoPURE II™ Plasmid Midiprep Kit (Zymo Research, Irvine, CA). Cell pellets were isolated by centrifugation from Eppendorf (Hamburg, Germany). Plasmid concentrations (ng μL -1 (range) was measured using a NanoDrop™ from Thermo Fischer Scientific (Waltham, MA). For the validation process, the GOI was cut using restriction enzymes and validated, followed by gel electrophoresis.

[0127] LC-MS / MS method for metabolite profiling: Metabolite standards were purchased from Sigma Aldrich (St. Louis, MO) and used for identification of waste inhibitor metabolites. Operating parameters and sample preparation protocols followed previously published protocols.

[0128] Cells, media and supplements: The CHO-K1 cell line expressing IgG (VRC01) antibody obtained from the National Institute of Health (NIH) was used in this study using two proprietary media. Cell inoculation and culture conditions followed previously published protocols.

[0129] Results and Discussion Pathway analysis of CHO metabolism. A comprehensive metabolomics analysis in different modes of CHO bioprocesses identified eight different growth by-products that accumulated throughout the entire culture period of the cells. However, although the effect of growth inhibition due to the presence of these metabolites in the CHO extracellular environment has been verified, the corresponding pathways and the extent to which their nutrient precursors are metabolized are currently unknown. Therefore, a strategic methodology was developed through pathway analysis combined with GEM to control the extent to which growth-inhibitory metabolites accumulate in the CHO bioprocess. Metabolic pathway analysis was performed for each metabolite through GEM, and the metabolites were then mapped onto the entire regulatory metabolic network. First, all metabolic enzymes that can either directly / indirectly catabolize toxic metabolites or redistribute metabolic flux to alternative metabolic branches that do not generate inhibitors as by-products were considered. The control of inhibitor accumulation by upregulating the expression of identified candidate genes involved in the regulation of such enzymes was evaluated. The list of candidate genes was further biologically validated by genetic manipulation studies for the evaluation of the resulting impact on growth. The final list of genes, the associated metabolic pathways, and the corresponding input metabolites, whose upregulation of enzyme activity can promote production for growth, are shown in Table 8.

[0130] Table 8: Genes, corresponding metabolic pathways and regulated downstream metabolites. Gene and pathway functionality were reported from previous studies studied in different mammalian cell lines. In this study, genes were inserted into plasmids and cells were cultured at 1 × 10 6 cells mL -1 The cells were transiently transfected when they reached a viable cell density of 100 μg / ml.

[0131] [Table 8]

[0132] 1 Enzymes control other metabolic fluxes that consume metabolites. 2 Downstream enzymes are involved in biochemical processes that utilize metabolites as input reactants. 3 It has been reported to be involved in metabolic pathways, but the exact relationship is under investigation.

[0133] Engineering strategies targeting metabolite accumulation. Pathway analysis performed on HICA and MSA revealed pathways derived from the catabolism of BCAAs. In the first step of catabolism, the transfer of an α-amino group from each of the precursor amino acids (leucine, isoleucine, and valine) produces three different respective branched-chain α-keto acids. The deamination of leucine is catalyzed by an amino acid transferase that transfers the amino group in leucine to an α-keto acid acceptor in α-ketoglutarate. Other BCAAs (e.g., valine and isoleucine) also share the same property of being an α-keto acid acceptor as leucine. Further analysis revealed that HICA is the end product of leucine metabolism, particularly through the direct conversion of keto acids to hydroxyl acids. Thus, regulation of the accumulation of α-ketoisocaproic acid (KIC) serves as a primary strategy in regulating metabolic flux to the formation of HICA, as shown in Figure 12. Specifically, by redistributing the flux that converts α-ketoglutarate to glutamate to other BCAAs, the presence of α-ketoglutarate that may be involved in the conversion of leucine to KIC is effectively reduced at a given time point. Thus, upregulation of cytoplasmic BCAA transaminase (BCAT1) is one effective approach to control HICA production. It could be argued that reducing KIC may compensate for the available flux toward the production of acetyl-CoA, a key substrate for energy production throughout the Krebs cycle. It is important to note that while this is still true regarding the amount of acetyl-CoA produced by leucine catabolism, acetyl-CoA can also be produced from isoleucine catabolism or from the conversion of succinyl-CoA, and thus downregulating the leucine degradation pathway will result in less HICA metabolites being produced as by-products.

[0134] Additional pathways were also studied via GEM. In CHO cells, alcohol dehydrogenase (ADH5) catalyzes an extracellular transport reaction involving formaldehyde and glutathionate. Study of glutathionate via GEM reveals key reactions that map glutathionate to the KIC via the leucine metabolic pathway involved in pathways 1 and 2, as shown in Figure 12. For pathway 1, glutathionate participates in the reaction that generates cysteine ​​from cystine, a by-product from the transport reaction that allocates cytoplasmic leucine to extracellular leucine. In pathway 2, glutathionate metabolism produces glutamate, which serves as a metabolite involved in the reaction that generates the KIC. Upregulation of ADH5 activity further metabolizes glutathione to formaglutathionate, reducing the amount of glutathione available to participate in other reactions that generate complementary substrates (reducing the amount of leucine produced that can be metabolized to KIC in pathway 1, or reducing the amount of glutamate that can catalyze reactions that generate KIC in pathway 2).

[0135] As mentioned earlier, tryptophan serves as the main substrate for nicotinamide metabolism, producing the metabolite TRI as a by-product (Figure 13A). Proline, as the main substrate for the TCA cycle, is mainly metabolized to pyruvate, but also produces NAP as a by-product (Figure 13B). Meanwhile, the metabolite TAA is a direct waste product resulting from the TCA cycle (Figure 13C). CMP is the main metabolite involved in pyrimidine metabolism (Figure 13D). Upregulation of CAT by metabolism of tryptophan and nicotinate / nicotinamide (target metabolite TRI), GOT1 and HOGA by metabolism of proline (target metabolite NAP), GOT1 by metabolism of glutamate and the TCA cycle (target metabolite TAA), and SLC35A1 by biosynthesis of N-glycans allows for control strategies for the accumulation of the identified metabolites.

[0136] Evaluation of plasmid transfection efficiency. The GFP gene was utilized as an expression reporter during translation to evaluate transfection efficiency. The plasmid engineered with the GFP gene was inserted into the CHO-S cell line, and the fluorescence intensity was evaluated by fluorescence microscopy and flow cytometry. The wild type condition was used as a control for the transfection condition. Both control and transfected cells were derived from the same culture to eliminate variations in growth cycle and other culture conditions. A comparison of the fluorescence intensity measurements at 2 and 3 days post-transfection for each of the transfection and control conditions is shown in Figure 14. The CHO-S cell line showed a transfection efficiency of 35.2% on day 2 with the medium supplied and the specific culture conditions (see Materials and Methods above). On day 3, the measured efficiency increased to 43.1%, demonstrating that the cells successfully expressed GFP during the CHO-S production period. As shown in Figure 15, the results were also confirmed by fluorescence microscopy imaging, since the cells also showed a high degree of fluorescence marker when visualized under fluorescence.

[0137] Quantification of expression levels of transfected genes of interest by qPCR. Expression levels of seven different genes transfected into CHO-S cells were assessed using relative quantification (RQ) compared to calibrators. The study was divided into eight unique sub-clone groups. Each sub-clone was transfected into cells at a plasmid concentration of 50 μg μL -1 Cells were transiently transfected at 100 ng / ml to overexpress the individual GOIs, and in an additional set of gene mix subclones, cells were transfected with all seven genes simultaneously but at lower individual plasmid concentrations than found in each case (6.25 μg μL per gene). -1 The total plasmid concentration for the seven genes was 43.75 μg μL -1). The study included a negative control in which no genetic engineering procedures, including plasmid transfection, were performed. Each subclone was cultured for 6 days. Cells from both control and transfected subclones were derived from the same culture to eliminate variations in growth cycles and other culture conditions. Plots of the RT-qPCR results are shown in Figure 16A-G. The level of gene expression in the control was the lowest in all cases, followed by the 50 μg μL plasmid concentration seen in each individual case. -1 6.25 μg μL per gene for each gene compared to -1 The total plasmid concentration was 43.75 μg μL for the seven genes. -1 It was revealed that the gene mix condition was followed by a gene mix condition with a RQ of 0.01 to 0.01. The range of RQ values ​​obtained showed considerable variation between each different transfected condition when compared against the gene mix condition, as well as for the control. Thus, this study suggests that although all engineered plasmids individually show different levels of gene expression when inserted into cells depending on the GOI, all show higher levels of gene expression compared to the negative control from samples obtained on day 3, when most of the gene expression in the batch culture of CHO cells occurs, providing a clear indication that the plasmids were successfully transfected into the cells and that all GOIs were expressed during the CHO translation process.

[0138] Gene functionality study by metabolic pathway analysis. As mentioned previously, HICA is produced as a downstream metabolite from the metabolism of leucine in mammalian cells, and its cellular biochemical activity is regulated by ADH5 and BCAT1 enzymes. A total of seven genes were transfected into the cells at a plasmid concentration of 50 μg μL -1 We transfected cells with all seven genes simultaneously, but at lower individual plasmid concentrations (6.25 μg μL per gene) than when each gene was transfected individually. -1 The total plasmid concentration of the seven genes was 43.75 μg μL. -1) was compared to a group of gene mix subclones transfected with 100-fold higher GFP-positive clones. The study included a negative control in which no genetic engineering work was performed, including transfection of plasmids. The results are shown in Figures 17A-G.

[0139] Adh5 and Bcat1 transfected clones showed 86.13% and 72.31% reduction in HICA accumulation, respectively (see FIG. 17A). With respect to branched chain amino acid (BCAA) metabolism, the isoleucine metabolic pathway actively interacts with the leucine metabolic pathway (see FIG. 12). By overexpressing Hadhb gene in CHO cells, this study showed that HICA accumulation in CHO was reduced by 27.05% compared to the control (FIG. 17C), suggesting that the cellular metabolic flux was more internally allocated to the metabolism of isoleucine and then less to the production of growth inhibitory by-products (in this case, HICA).

[0140] With respect to TRI, the metabolite is part of the metabolism of nicotinamide and is generated as a downstream product from the metabolism of NAD and NADH, of which nicotinamide is the upstream precursor. In this study, clones overexpressing Cat and Got1 genes showed a 27.8% and 40.43% decrease in the accumulation of TR1, respectively (see FIG. 17B). Furthermore, clones overexpressing Got1, Hoga1 and Slc35a1 genes showed a 35.68%, 33.59% and 50.63% decrease in the accumulation of CMP, respectively (see FIG. 17C). Thus, the data suggest that cells reallocated internal metabolic flux towards the metabolism of glutamine, pyrimidine, arginine and proline, and less towards the production of growth inhibitory by-products (in this case, CMP).

[0141] Overall, these results indicate that HICA, TRI, and CMP are metabolic by-products generated through the metabolism of BCAAs, tryptophan, glutamine, and pyrimidines in the CHO system, as in other mammalian host cells. Furthermore, through genetic engineering studies to overexpress upstream genes involved in different metabolic pathways, the studies also showed that upregulation of the translational activity of metabolic enzymes, including ADH5, BCAT1, CAT, GOT1, HOGA1, and SLC35A1, could control the production of growth-inhibiting metabolites by either increasing the flux of biochemical reactions that metabolize waste by-products (enzymes downstream to waste generation) or by reallocating available metabolic flux to other alternative metabolic pathways.

[0142] Interestingly, the accumulation of HICA in CHO cultures showed a significant decrease when the pyrimidine transporter gene Slc35a1 was overexpressed (73.51% less accumulation compared to the control, see Figure 17A). Furthermore, upregulation of Bcat1 and Hadhb1 also reduced the accumulation of TRI by 42.96% and 35.38%, respectively. Thus, the data suggest that SLC35A1, BCAT1, and HADHB1 enzymes may interact with the production of HICA and TRI through understudied metabolic pathways. The results also showed that in most cases, overexpression of metabolic genes reduced the accumulation of CMP when compared to the control, suggesting that pyrimidines interact with many different metabolic pathways of amino acids.

[0143] The results also revealed that the accumulation of TAA, MSA, and NAP concentrations at harvest in all subclones showed no significant difference when compared to the control (see Figure 17D, E, and F). Without being limited by theory, it is hypothesized that these metabolites are not derived from reported metabolic pathways, as in other mammalian cell lines (see Table 7).

[0144] Thus, genetic intervention of the corresponding genes responsible for glycolysis and / or the proline and isoleucine metabolic pathways could possibly provide a more robust set of tools to develop appropriate control strategies and manipulate the production of these growth-inhibitory metabolites.

[0145] Evaluation of culture performance. Growth profiles of all individual subclones are shown in Figure 18A-B. In addition, 6.25 μg μL per gene -1 The effect of upregulation of all engineered vectors (7 genes) transfected into the CHO-S cell line at a concentration of 0.01 mg / ml was also studied (see Figure 19). In both cases, cells were cultivated in batch mode until cell viability dropped below 80%. Overall, all subclones (individual genes as well as gene mixes) with overexpression of metabolic genes showed enhanced growth when compared to the control. Overall, these results suggest that the growth of CHO cells in bioprocesses can be improved either by increasing the generation of cellular energy utilized for growth by metabolizing growth-inhibitory metabolites as reactants in biochemical pathways that have been poorly studied (thus reducing their accumulated concentration) or by redistributing metabolic flux to alternative metabolic pathways that do not generate metabolic by-products that may inhibit cell growth.

[0146] Nutrient consumption profiles. Nutrient consumption profiles of all transfected subclones are shown in Figure 20A-G. Overall, cells across all conditions consumed the highest amounts of nutrients during day 4 of batch culture. Overall, the consumption profiles of key exchangeable ions including sodium and potassium from the transfected subclones were generally higher when compared to the control. Traditionally, cells consume less Na than Na. + / K +It utilizes pumping mechanisms to regulate water balance and other cellular physiological states. It is speculated that a faster rate of ion uptake would drive the activity of the major nutrient exchange gradient, suggesting that cells would exhibit more rapid substrate utilization and metabolic activity rates when the major target biochemical pathways that could directly / indirectly catabolize the identified metabolites are upregulated. This further translates into higher accumulation profiles of ammonia and lactate as measured on days 4 and 6 of the batch process (see Figures 20C and 20D). However, lower peak viable cell density (VCD) and cumulative integrated viable cell density (IVCD) across all conditions were not observed, as exemplified in Figures 18A-B, where all engineered subclones show better culture performance compared to the control. Interestingly, all transfected subclones showed higher glucose consumption profiles throughout the culture period (less glucose accumulated, see Figure 20D), and even more aggressive consumption profiles from days 4 to 6. Comparing the nutrient consumption profiles (see Figure 20A-G), this culture period coincides with the production phase of the cells, thus allowing the cells to utilize more glucose for protein production, due to the improved cell growth performance (higher cell density). The results obtained from the different cell activity profiles suggest that the glucose consumption profile is higher in the production phase, as the cells mainly utilize glucose to generate glycans, and the cell productivity is also upregulated. Future studies can be performed addressing the titer profile of the cells after transfection to analyze the productivity of the CHO subclones with improved growth while producing less toxic growth inhibitors. Interestingly, the concentration of glutamine measured on day 4 from the Got1 subclone is significantly higher compared to the control. Traditionally, the GOT1 enzyme catalyzes the biosynthesis of glutamate from aspartate and cysteine. It can be speculated that the upregulation of Got1 induces the production of more glutamate, which can be converted to glutamine via the glutamine synthesis pathway regulated by glutamine synthetase.Nevertheless, future studies on genetic intervention of the studied genes involved in these metabolic pathways are needed to better explain the observed trends in nutrient exchange profiles.

[0147] The nutrient consumption profile of the gene mix condition was also studied, as shown in Figures 21A-G. Overall, the accumulated concentrations of sodium and potassium measured throughout the culture period were higher in the gene mix condition when compared to the control condition, except at day 7, when the sodium and potassium accumulation profile of the control condition was higher (see Figures 21A and 21B). The data are consistent with the trends observed from the growth profiles, where the gene mix subclones achieved higher peak VCD and cumulative IVCD. It was speculated that the higher cell density from the gene mix cultures consumed more sodium and potassium to maintain growth and other cellular activities, except up until day 7, when cell death occurred. Interestingly, the gene mix condition consumed less glutamine and glutamate (higher accumulation of glutamine and glutamate measured in the spent medium, Figures 21E and 21G), while consuming less ammonia and lactate (lower concentrations of ammonia and lactate measured, as seen in Figures 21C and 21D), and maintained a higher peak VCD and a higher overall cumulative IVCD (Figures 19A and 19B). The data suggests that when all seven genes were transfected into cells simultaneously, the effects of the transfected genes were more significant and consumed less nutrients under the conditions studied, allowing them to produce less toxic growth by-products and inhibitory metabolites (see FIG. 17), enabling them to achieve higher overall cell densities and better culture performance.

[0148] conclusion Mammalian cells are known to secrete growth-inhibitory metabolites during the growth and production phases, which can interfere with cell performance and negatively affect final productivity and product quality attributes. Eight growth inhibitors have been identified in typical CHO and HEK293 bioprocesses. Seven of these genes (Adh5, Bcat1, Cat, Got1, Hadhb, Hoga1, and Slc35a1) were transfected into CHO cells to modulate their metabolism. The concentrations of metabolites, measured by LC-MS / MS, were successfully reduced by re-allocating the intracellular metabolic fluxes entering key metabolic pathways capable of direct or indirect metabolism of the previously identified inhibitors. The seven identified genes were analyzed using a 50-mL HPLC system at 8 μg μL per gene. -1 When cells were individually transfected with plasmid concentrations of 1000 μg / ml, the peak cellular VCD and cumulative IVCD were improved compared to the control throughout the entire culture period after transfection. The concentrations of inhibitory metabolites accumulated in the medium after consumption of each subclone were also reduced, as measured by LC-MS / MS.

[0149] Each of the seven genes was tested at 6.25 μg μL per gene. -1 The cells were co-transfected with 100 μg of each gene (a total of 43.75 μg μL -1of plasmid concentration). The results showed that improved cell performance was also achieved with the gene mix achieving higher peak VCD and cumulative IVCD. Interestingly, the effect of multiple gene transfection seemed to have a more pronounced impact on cell metabolism compared to single gene transfection. The study revealed that the gene mix condition resulted in less glutamine and glutamate being consumed (more accumulation of glutamine and glutamate was measured in the spent medium), which allowed the cells to secrete less ammonia and lactate while at the same time maintaining a better growth profile and producing fewer growth inhibitory metabolites. Overall, the study shows that by combining genome-scale modeling with pathway analysis, the study successfully uncovered a control strategy platform that can mitigate metabolite accumulation by allocating metabolic fluxes to specific metabolic pathways that can metabolize growth inhibitors and promote cell growth.

[0150] Use of the terms "a" and "an" and "the" and similar references (particularly in the context of the following claims) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Terms such as first, second, etc., as used herein are not meant to indicate a particular order, but are merely meant for convenience, e.g., to indicate multiple layers. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. Recitations of ranges of values ​​are intended only to serve as a shorthand method of individually referring to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually recited herein. The endpoints of all ranges are included within the ranges and can be independently combined. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") is intended merely to better describe 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.

[0151] Although the present invention has been described with reference to exemplary embodiments, those skilled in the art will recognize that various changes can be made and equivalents can be substituted for the elements thereof without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope of the invention. It is therefore intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but will include all embodiments that fall within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. A method of cell culture, the method comprising: (i) culturing cells in a cell culture medium; and (ii) maintaining at least one metabolite below an inhibitory concentration in the cell culture medium for the at least one metabolite by modulating enzyme expression to reduce synthesis of the at least one metabolite; the at least one metabolite is aconitic acid (AA), leucine acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI), N-acetylputresinium (NAP), or a combination thereof; The method, wherein the enzyme comprises ADH5, BCAT1, CAT, GOT1, HADHB, HOGA1, SLC35A1, or a combination thereof.

2. 2. The method of claim 1, further comprising: measuring the concentration of the at least one metabolite; and modulating enzyme expression to decrease the synthesis of the metabolite when the measured concentration of the at least one metabolite is above a predetermined value.

3. 3. The method of claim 2, wherein modulating enzyme expression to decrease said synthesis of said metabolite comprises adding a recombinant DNA molecule to overexpress an enzyme, said enzyme being a downstream enzyme.

4. the metabolite is HICA and the enzyme comprises ADH5, BCAT1, SLC35A1, GOT1, HADHB, or a combination thereof; the metabolite is CMP and the enzyme comprises ADH5, BCAT1, CAT, GOT1, HOGA1, SLC35A1, HADHB, or a combination thereof; the metabolite is MSA and the enzyme comprises BCAT1, GOT1, HADHB, HOGA1, SLC35A1, or a combination thereof; the metabolite is TRI and the enzyme comprises BCAT1, GOT1, HADHB, CAT, HOGA1, SLC35A1, or a combination thereof; the metabolite is NAP and the enzyme comprises BCAT1, SLC35A1, HOGA1, GOT1, CAT, HADHB, or a combination thereof; the metabolite is TAA and the enzyme is BCAT1, ADH5, GOT1, HOGA1, SLC35A1, HADHB, or a combination thereof; or The method of claim 1 , comprising:

5. 2. The method of claim 1, wherein the enzymes include ADH5, BCAT1, GOT1, HADHB, HOGA1, SLC35A1, and CAT.

6. 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.

7. 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.

8. 2. The method of claim 1, wherein the cell is a CHO cell, a HEK293 cell, a HT-1080 cell, an engineered T cell, or an engineered natural killer cell.

9. 2. The method of claim 1, wherein the cell culture is a batch culture, a fed-batch culture, or a perfusion culture.

10. The method of claim 1 , wherein the cell expresses a recombinant protein, gene product, or cell product.

11. The method of claim 10, wherein the recombinant protein is a monoclonal antibody.

12. 2. The method of claim 1, wherein cell growth 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).

13. 13. The method of claim 12, wherein the cell growth is determined by maximum viable cell density and is increased by at least 5% compared to the control culture.

14. the metabolite is AA and the concentration of AA in the cell culture medium is less than 440 μM; the metabolite is HICA and the concentration of HICA in the cell culture medium is less than 23.5 μM; the metabolite is CMP and the concentration of CMP in the cell culture medium is less than 10 μM; the metabolite is MSA and the concentration of MSA in the cell culture medium is less than 3.75 μM; the metabolite is TRI and the concentration of TRI in the cell culture medium is less than 0.35 μM; or the metabolite is NAP and the concentration of NAP in the cell culture medium is less than 0.3 μM; The method of claim 1.

15. 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.

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