Cell selection method and cell metabolism modification method

Abstract

This invention provides a method for identifying, selecting, or culturing cells containing a target nucleic acid sequence. [Solution] A glutamine synthase knockout (GS-KO) cell is provided, comprising a nucleic acid comprising a sequence encoding a heterologous lipid metabolism modifier (LMM) operably linked to a nucleic acid comprising a control region, wherein the nucleic acid comprising the sequence encoding the heterologous LMM is arranged in an integrated vector, and the integrated vector further comprises a nucleic acid comprising a sequence encoding a selectable marker, if present, that enables survival or growth in a tyrosine-deficient medium, wherein the marker comprises an enzyme that catalyzes the rate-limiting step of the tyrosine biosynthesis pathway. Furthermore, a method for culturing GS-KO CHO cells comprising the LMM is provided.

Description

[Technical Field]

[0001] This disclosure relates to methods and compositions for identifying, selecting, or culturing cells containing a target nucleic acid sequence. [Background technology]

[0002] Eukaryotic cells, which are nutrient-dependent for essential compounds necessary for growth, can utilize the expression of heterologous products by coupling the expression of their products with the expression of ectopic enzymes that enable the synthesis of these essential compounds. For example, mutant mouse cells that are thymidine-dependent cannot grow in the absence of externally supplied thymidine (Ayusawa et al. (1981) Somatic Cell Genet. 7(5):523~534). Revertant mutants with restored thymidylate synthase activity were able to grow under thymidine-depleted conditions.

[0003] Mammalian cell expression systems are commonly used for the production of recombinant biological products, such as therapeutic biologics. However, large-scale production of such products involves high initial costs due to the difficulty in efficiently generating stable cell lines that efficiently express large quantities of the desired product. Therefore, there is a need in the art for improvements in methods and compositions for producing cells that can be used to produce recombinant biological products.

[0004] [Summary of the Invention] Compositions and methods for identifying, selecting, or culturing cells containing a target nucleic acid sequence (e.g., one or more target nucleic acid sequences) are described herein. Generally, a nucleic acid containing the target nucleic acid and a sequence encoding an enzyme molecule involved in amino acid biosynthesis is introduced into cells. The cells are then grown in an amino acid-deficient medium so that the cells containing the introduced nucleic acid can grow. In some cases, the cells further contain an inhibitor of the enzyme molecule to increase the rigor of selection. Nutritional requirements for compounds other than amino acids (e.g., trace metals, small molecules, nucleic acids, or other metabolites known in the art) are intended to be used in the selection methods described herein.

[0005] In one aspect, the present invention relates to a method for identifying, selecting, or culturing cells containing a target nucleic acid sequence, a)(i) Target nucleic acid sequence and (ii) When expressed, nucleic acid sequences that result in high levels of activity of enzymes in amino acid synthesis pathways, such as the proline synthesis pathway, for example, nucleic acid sequences that encode enzyme molecules containing activity A step of preparing cells containing nucleic acids, such as vectors, such as replicable vectors or integration vectors, b) A step of culturing cells containing nucleic acid sequences in a medium containing amino acids, such as proline, at levels insufficient to support the growth of cells that are the same as cells that do not have high activity, under conditions sufficient to allow the growth of cells containing nucleic acid sequences. This invention includes a method for identifying, selecting, or culturing cells containing heterogeneous nucleic acid sequences. In one aspect, the present invention relates to a method for identifying, selecting, or culturing cells containing a target nucleic acid sequence, a)(i) Target nucleic acid sequence and (ii) When expressed, nucleic acid sequences that result in high levels of activity of enzymes in amino acid synthesis pathways, such as the proline synthesis pathway, for example, nucleic acid sequences that encode enzyme molecules containing activity The steps include: preparing cells containing nucleic acids, such as vectors, such as replicable vectors; b) A step of culturing cells containing nucleic acid sequences in a medium (and optionally containing enzyme inhibitors) that has sufficient conditions to enable the growth of cells containing nucleic acid sequences, but insufficient levels of amino acids, such as proline, to support the growth of cells that are the same as the non-highly active target cells, c) A step of culturing cells containing a nucleic acid sequence in the presence of a second medium (and optionally a medium containing an inhibitor of the second enzyme) having a second amino acid, such as tyrosine, at a level insufficient to support the growth of cells that are the same as the non-highly active target cells, under conditions sufficient to enable the growth of the nucleic acid sequence-containing cells. This invention includes a method for identifying, selecting, or culturing cells containing heterogeneous nucleic acid sequences.

[0006] In one embodiment, the present invention is characterized by a cell comprising a sequence including a regulatory region derived from any of the following: an SV40 promoter sequence, an mCMV promoter sequence, or a PGK promoter sequence, for example, a sequence encoding a heterologous lipid metabolism modifier (LMM) operably linked to a promoter sequence. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 is a schematic diagram showing the rate-limiting step in the mammalian proline synthesis pathway. Question marks indicate potential inhibitors that may be used to inhibit the conversion of glutamate to glutamate 5-semialdehyde. [Figure 2] Figure 2 shows plasmid maps of exemplary vectors containing the P5CS and eGFP genes. [Figure 3A] Figures 3A and 3B are a series of graphs showing the growth profiles of GSKO cells cultured in various media (complete medium (6 mM glutamine), glutamine-deficient medium (without glutamine), and proline-deficient medium (without proline)). Figure 3A shows the viable cell concentration, and Figure 3B shows the culture viability. [Figure 3B]Figures 3A-3B are a series of graphs showing the growth profiles of GSKO cells cultured in various media (complete medium (6 mM glutamine), glutamine-deficient medium (without glutamine), and proline-deficient medium (without proline)). Figure 3A shows the viable cell concentration, and Figure 3B shows the culture viability. [Figure 4A] Figures 4A-4C are a series of graphs showing the plasmid maps of exemplary vectors containing the etanercept gene (Figure 4A), glutamine synthetase (GS: glutamine synthetase) and eGFP gene (Figure 4B), or P5CS and eGFP gene (Figure 4C). [Figure 4B] Figures 4A-4C are a series of graphs showing the plasmid maps of exemplary vectors containing the etanercept gene (Figure 4A), glutamine synthetase (GS: glutamine synthetase) and eGFP gene (Figure 4B), or P5CS and eGFP gene (Figure 4C). [Figure 4C] Figures 4A-4C are a series of graphs showing the plasmid maps of exemplary vectors containing the etanercept gene (Figure 4A), glutamine synthetase (GS: glutamine synthetase) and eGFP gene (Figure 4B), or P5CS and eGFP gene (Figure 4C). [Figure 5A] Figures 5A-5D are a series of graphs showing the effects of cell cultures transiently transfected with one of the exemplary vectors shown in Figure 4 and then cultured in CD-CHO (supplemented to contain 6 mM L-glutamine), tyrosine-free (without tyrosine), or proline-free (without proline) medium. The viable cell concentration (Figure 5A), culture viability (Figure 5B), average eGFP fluorescence signal (Figure 5C), and percentage of cells exceeding a predetermined eGFP fluorescence intensity threshold (Figure 5D) at 72 hours and 168 hours after transfection are shown. Error bars represent the standard deviation of the mean (n = 3). [Figure 5B]Figures 5A-5D are a series of graphs showing the effects of cell cultures transiently transfected with one of the exemplary vectors shown in Figure 4 and then cultured in CD-CHO (supplemented to contain 6 mM L-glutamine), tyrosine-free or proline-free media. The viable cell concentration (Figure 5A), culture viability (Figure 5B), average eGFP fluorescence signal (Figure 5C), and percentage of cells exceeding a predetermined eGFP fluorescence intensity threshold (Figure 5D) at 72 hours and 168 hours post-transfection are shown. Error bars represent the standard deviation of the mean (n = 3). [Figure 5C] Figures 5A-5D are a series of graphs showing the effects of cell cultures transiently transfected with one of the exemplary vectors shown in Figure 4 and then cultured in CD-CHO (supplemented to contain 6 mM L-glutamine), tyrosine-free or proline-free media. The viable cell concentration (Figure 5A), culture viability (Figure 5B), average eGFP fluorescence signal (Figure 5C), and percentage of cells exceeding a predetermined eGFP fluorescence intensity threshold (Figure 5D) at 72 hours and 168 hours post-transfection are shown. Error bars represent the standard deviation of the mean (n = 3). [Figure 5D] Figures 5A-5D are a series of graphs showing the effects of cell cultures transiently transfected with one of the exemplary vectors shown in Figure 4 and then cultured in CD-CHO (supplemented to contain 6 mM L-glutamine), tyrosine-free or proline-free media. The viable cell concentration (Figure 5A), culture viability (Figure 5B), average eGFP fluorescence signal (Figure 5C), and percentage of cells exceeding a predetermined eGFP fluorescence intensity threshold (Figure 5D) at 72 hours and 168 hours post-transfection are shown. Error bars represent the standard deviation of the mean (n = 3). [Figure 6A]Figures 6A–6C are a series of figures showing the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline. Figure 6A shows a histogram obtained using flow cytometry analysis. Figures 6B and 6C, respectively, show fluorescence images generated by confocal microscopy; the channels shown are DAPI, P5CS:TRITC, and eGFP. Figure 6C shows a comparison between the P5CS-generating pool and the GSKO host. [Figure 6B] Figures 6A–6C are a series of figures showing the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline. Figure 6A shows a histogram obtained using flow cytometry analysis. Figures 6B and 6C, respectively, show fluorescence images generated by confocal microscopy; the channels shown are DAPI, P5CS:TRITC, and eGFP. Figure 6C shows a comparison between the P5CS-generating pool and the GSKO host. [Figure 6C] Figures 6A–6C are a series of figures showing the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline. Figure 6A shows a histogram obtained using flow cytometry analysis. Figures 6B and 6C, respectively, show fluorescence images generated by confocal microscopy; the channels shown are DAPI, P5CS:TRITC, and eGFP. Figure 6C shows a comparison between the P5CS-generating pool and the GSKO host. [Figure 7] Figure 7 shows a series of Western blot images illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline. The vectors used to construct the cell pools contained either the eGFP gene (eGFP), the SCD1 gene whose expression is driven by the SV40 promoter (SV40 SCD1), or the SCD1 gene whose expression is driven by the mCMV promoter (mCMV SCD1). Western blot analysis was performed to identify the expression of P5CS, TAT, and SCD1 (the target gene). [Figure 8A]Figures 8A–8F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. L-azetidine-2-carboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 8A) and culture viability (Figure 8B). Cells were also analyzed for the mean culture fluorescence (Figure 8C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 8D) and 103 (Figure 8E). Figure 8F shows Western blot analysis of solubilized saturations collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 8B] Figures 8A–8F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. L-azetidine-2-carboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 8A) and culture viability (Figure 8B). Cells were also analyzed for the mean culture fluorescence (Figure 8C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 8D) and 103 (Figure 8E). Figure 8F shows Western blot analysis of solubilized saturations collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 8C]Figures 8A–8F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. L-azetidine-2-carboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 8A) and culture viability (Figure 8B). Cells were also analyzed for the mean culture fluorescence (Figure 8C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 8D) and 103 (Figure 8E). Figure 8F shows Western blot analysis of solubilized saturations collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 8D] Figures 8A–8F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. L-azetidine-2-carboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 8A) and culture viability (Figure 8B). Cells were also analyzed for the mean culture fluorescence (Figure 8C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 8D) and 103 (Figure 8E). Figure 8F shows Western blot analysis of solubilized saturations collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 8E]Figures 8A–8F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. L-azetidine-2-carboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 8A) and culture viability (Figure 8B). Cells were also analyzed for the mean culture fluorescence (Figure 8C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 8D) and 103 (Figure 8E). Figure 8F shows Western blot analysis of solubilized saturations collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 8F] Figures 8A–8F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. L-azetidine-2-carboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 8A) and culture viability (Figure 8B). Cells were also analyzed for the mean culture fluorescence (Figure 8C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 8D) and 103 (Figure 8E). Figure 8F shows Western blot analysis of solubilized saturations collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 9A]Figures 9A–9F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of 3,4-dehydro-L-proline, a P5CS inhibitor. Cells were cultured in 96-well deep plates for 9 days after being added at various different concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 9A) and culture viability (Figure 9B). Cells were also analyzed for the mean culture fluorescence (Figure 9C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 9D) and 103 (Figure 9E). Figure 9F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 9B] Figures 9A–9F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of 3,4-dehydro-L-proline, a P5CS inhibitor. Cells were cultured in 96-well deep plates for 9 days after being added at various different concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 9A) and culture viability (Figure 9B). Cells were also analyzed for the mean culture fluorescence (Figure 9C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 9D) and 103 (Figure 9E). Figure 9F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 9C]Figures 9A–9F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of 3,4-dehydro-L-proline, a P5CS inhibitor. Cells were cultured in 96-well deep plates for 9 days after being added at various different concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 9A) and culture viability (Figure 9B). Cells were also analyzed for the mean culture fluorescence (Figure 9C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 9D) and 103 (Figure 9E). Figure 9F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 9D] Figures 9A–9F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of 3,4-dehydro-L-proline, a P5CS inhibitor. Cells were cultured in 96-well deep plates for 9 days after being added at various different concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 9A) and culture viability (Figure 9B). Cells were also analyzed for the mean culture fluorescence (Figure 9C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 9D) and 103 (Figure 9E). Figure 9F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 9E]Figures 9A–9F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of 3,4-dehydro-L-proline, a P5CS inhibitor. Cells were cultured in 96-well deep plates for 9 days after being added at various different concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 9A) and culture viability (Figure 9B). Cells were also analyzed for the mean culture fluorescence (Figure 9C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 9D) and 103 (Figure 9E). Figure 9F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 9F] Figures 9A–9F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of 3,4-dehydro-L-proline, a P5CS inhibitor. Cells were cultured in 96-well deep plates for 9 days after being added at various different concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 9A) and culture viability (Figure 9B). Cells were also analyzed for the mean culture fluorescence (Figure 9C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 9D) and 103 (Figure 9E). Figure 9F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 10A]Figures 10A–10F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-4-thiazolidinecarboxylic acid, a P5CS inhibitor. L-4-thiazolidinecarboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 10A) and culture viability (Figure 10B). Cells were also analyzed for the mean culture fluorescence (Figure 10C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 10D) and 103 (Figure 10E). Figure 10F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 10B] Figures 10A–10F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-4-thiazolidinecarboxylic acid, a P5CS inhibitor. L-4-thiazolidinecarboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 10A) and culture viability (Figure 10B). Cells were also analyzed for the mean culture fluorescence (Figure 10C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 10D) and 103 (Figure 10E). Figure 10F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 10C]Figures 10A–10F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-4-thiazolidinecarboxylic acid, a P5CS inhibitor. L-4-thiazolidinecarboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 10A) and culture viability (Figure 10B). Cells were also analyzed for the mean culture fluorescence (Figure 10C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 10D) and 103 (Figure 10E). Figure 10F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 10D] Figures 10A–10F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-4-thiazolidinecarboxylic acid, a P5CS inhibitor. L-4-thiazolidinecarboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 10A) and culture viability (Figure 10B). Cells were also analyzed for the mean culture fluorescence (Figure 10C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 10D) and 103 (Figure 10E). Figure 10F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 10E]Figures 10A–10F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-4-thiazolidinecarboxylic acid, a P5CS inhibitor. L-4-thiazolidinecarboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 10A) and culture viability (Figure 10B). Cells were also analyzed for the mean culture fluorescence (Figure 10C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 10D) and 103 (Figure 10E). Figure 10F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 10F] Figures 10A–10F show a series of diagrams illustrating cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-4-thiazolidinecarboxylic acid, a P5CS inhibitor. L-4-thiazolidinecarboxylic acid was added at various different concentrations, and the cells were cultured in 96-well deep plates for 9 days. These cells were analyzed for cell growth by measuring viable cell concentration (Figure 10A) and culture viability (Figure 10B). Cells were also analyzed for the mean culture fluorescence (Figure 10C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 10D) and 103 (Figure 10E). Figure 10F shows Western blot analysis of solubilized saturates collected at specified time points and probed to highlight P5CS, β-actin, and eGFP proteins. [Figure 11A]Figures 11A–11E show a series of figures illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. Cells were cultured for 9 days in 24-well static plates with L-azetidine-2-carboxylic acid added at various concentrations. Cell growth was analyzed by measuring viable cell concentration (Figure 11A) and culture viability (Figure 11B). Cells were also analyzed for the average culture fluorescence value (Figure 11C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 11D) and 103 (Figure 11E). [Figure 11B] Figures 11A–11E show a series of figures illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. Cells were cultured for 9 days in 24-well static plates with L-azetidine-2-carboxylic acid added at various concentrations. Cell growth was analyzed by measuring viable cell concentration (Figure 11A) and culture viability (Figure 11B). Cells were also analyzed for the average culture fluorescence value (Figure 11C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 11D) and 103 (Figure 11E). [Figure 11C] Figures 11A–11E show a series of figures illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. Cells were cultured for 9 days in 24-well static plates with L-azetidine-2-carboxylic acid added at various concentrations. Cell growth was analyzed by measuring viable cell concentration (Figure 11A) and culture viability (Figure 11B). Cells were also analyzed for the average culture fluorescence value (Figure 11C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 11D) and 103 (Figure 11E). [Figure 11D]Figures 11A–11E show a series of figures illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. Cells were cultured for 9 days in 24-well static plates with L-azetidine-2-carboxylic acid added at various concentrations. Cell growth was analyzed by measuring viable cell concentration (Figure 11A) and culture viability (Figure 11B). Cells were also analyzed for the average culture fluorescence value (Figure 11C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 11D) and 103 (Figure 11E). [Figure 11E] Figures 11A–11E show a series of figures illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of L-azetidine-2-carboxylic acid, a P5CS inhibitor. Cells were cultured for 9 days in 24-well static plates with L-azetidine-2-carboxylic acid added at various concentrations. Cell growth was analyzed by measuring viable cell concentration (Figure 11A) and culture viability (Figure 11B). Cells were also analyzed for the average culture fluorescence value (Figure 11C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 11D) and 103 (Figure 11E). [Figure 12A] Figures 12A–12E show a series of figures illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of the P5CS inhibitor 3,4-dehydro-L-proline. Cells were cultured in 24-well static plates for 9 days after being added at various concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 12A) and culture viability (Figure 12B). Cells were also analyzed for the mean fluorescence value of the culture (Figure 12C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 12D) and 103 (Figure 12E). [Figure 12B]Figures 12A–12E show a series of figures illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of the P5CS inhibitor 3,4-dehydro-L-proline. Cells were cultured in 24-well static plates for 9 days after being added at various concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 12A) and culture viability (Figure 12B). Cells were also analyzed for the mean fluorescence value of the culture (Figure 12C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 12D) and 103 (Figure 12E). [Figure 12C] Figures 12A–12E show a series of figures illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of the P5CS inhibitor 3,4-dehydro-L-proline. Cells were cultured in 24-well static plates for 9 days after being added at various concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 12A) and culture viability (Figure 12B). Cells were also analyzed for the mean fluorescence value of the culture (Figure 12C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 12D) and 103 (Figure 12E). [Figure 12D] Figures 12A–12E show a series of figures illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of the P5CS inhibitor 3,4-dehydro-L-proline. Cells were cultured in 24-well static plates for 9 days after being added at various concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 12A) and culture viability (Figure 12B). Cells were also analyzed for the mean fluorescence value of the culture (Figure 12C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 12D) and 103 (Figure 12E). [Figure 12E]Figures 12A–12E show a series of figures illustrating the analysis of cell pools generated using P5CS overexpression and cultured in the absence of proline and in the presence of the P5CS inhibitor 3,4-dehydro-L-proline. Cells were cultured in 24-well static plates for 9 days after being added at various concentrations of 3,4-dehydro-L-proline. Cell growth was analyzed by measuring viable cell concentration (Figure 12A) and culture viability (Figure 12B). Cells were also analyzed for the mean fluorescence value of the culture (Figure 12C) and the percentage of cells exceeding predetermined thresholds for 102 (Figure 12D) and 103 (Figure 12E). [Figure 13] Figure 13 shows the recovery rate (in days) of cultures cotransfected with an exemplary vector containing an exemplary lipid metabolism modifier (LMM) gene (SCD1, SREBF1, or SREB411) under the control of an exemplary promoter (SV40, mCMV, or PGK promoter), and a second exemplary vector containing a gene encoding an exemplary recombinant protein of interest (etanercept, cergutuzumab, infliximab, or cB72.3). [Figure 14A] Figures 14A–14D are a series of graphs showing the mean viable cell concentration (VCC) at 10⁶ / ml of cells transfected with the indicated LMM genes under the control of the indicated promoters, and with genes encoding etanercept (Figure 14A), cergutuzumab (Figure 14B), infliximab (Figure 14C), or cB72.3 (Figure 14D). Cell cultures were grown in deep-well plates, and VCC was measured by Celigo cell counting for each culture / well. [Figure 14B]Figures 14A–14D are a series of graphs showing the mean viable cell concentration (VCC) at 10⁶ / ml of cells transfected with the indicated LMM genes under the control of the indicated promoters, and with genes encoding etanercept (Figure 14A), cergutuzumab (Figure 14B), infliximab (Figure 14C), or cB72.3 (Figure 14D). Cell cultures were grown in deep-well plates, and VCC was measured by Celigo cell counting for each culture / well. [Figure 14C] Figures 14A–14D are a series of graphs showing the mean viable cell concentration (VCC) at 10⁶ / ml of cells transfected with the indicated LMM genes under the control of the indicated promoters, and with genes encoding etanercept (Figure 14A), cergutuzumab (Figure 14B), infliximab (Figure 14C), or cB72.3 (Figure 14D). Cell cultures were grown in deep-well plates, and VCC was measured by Celigo cell counting for each culture / well. [Figure 14D] Figures 14A–14D are a series of graphs showing the mean viable cell concentration (VCC) at 10⁶ / ml of cells transfected with the indicated LMM genes under the control of the indicated promoters, and with genes encoding etanercept (Figure 14A), cergutuzumab (Figure 14B), infliximab (Figure 14C), or cB72.3 (Figure 14D). Cell cultures were grown in deep-well plates, and VCC was measured by Celigo cell counting for each culture / well. [Figure 15A]Figures 15A–15D are a series of graphs showing the mean viable cell concentration (VCC) at 10⁶ / ml of cells during abridged fed-batch overgrow (aFOG). Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 15A), cergutuzumab (Figure 15B), infliximab (Figure 15C), or cB72.3 (Figure 15D). Cell cultures were grown in deep-well plates, and VCC was measured by Celigo cell counting for each culture / well. [Figure 15B] Figures 15A–15D are a series of graphs showing the mean viable cell concentration (VCC) at 10⁶ / ml of cells during abridged fed-batch overgrow (aFOG). Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 15A), cergutuzumab (Figure 15B), infliximab (Figure 15C), or cB72.3 (Figure 15D). Cell cultures were grown in deep-well plates, and VCC was measured by Celigo cell counting for each culture / well. [Figure 15C] Figures 15A–15D are a series of graphs showing the mean viable cell concentration (VCC) at 10⁶ / ml of cells during abridged fed-batch overgrow (aFOG). Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 15A), cergutuzumab (Figure 15B), infliximab (Figure 15C), or cB72.3 (Figure 15D). Cell cultures were grown in deep-well plates, and VCC was measured by Celigo cell counting for each culture / well. [Figure 15D]Figures 15A–15D are a series of graphs showing the mean viable cell concentration (VCC) at 10⁶ / ml of cells during abridged fed-batch overgrow (aFOG). Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 15A), cergutuzumab (Figure 15B), infliximab (Figure 15C), or cB72.3 (Figure 15D). Cell cultures were grown in deep-well plates, and VCC was measured by Celigo cell counting for each culture / well. [Figure 16A] Figures 16A–16D are a series of graphs showing bubble plots of specific productivity of cell cultures expressing the LMM gene and the gene of interest in aFOG. Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 16A), cergutuzumab (Figure 16B), infliximab (Figure 16C), or cB72.3 (Figure 16D). Cultures were grown in deep-well plates, and VCC was measured by Celigo cell count for each culture / well. Productivity was measured using an Octet instrument with a protein A biosensor. Each indicated data point corresponds to an individual culture; the diameter of each data point indicates the product concentration achieved for that culture. [Figure 16B] Figures 16A–16D are a series of graphs showing bubble plots of specific productivity of cell cultures expressing the LMM gene and the gene of interest in aFOG. Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 16A), cergutuzumab (Figure 16B), infliximab (Figure 16C), or cB72.3 (Figure 16D). Cultures were grown in deep-well plates, and VCC was measured by Celigo cell count for each culture / well. Productivity was measured using an Octet instrument with a protein A biosensor. Each indicated data point corresponds to an individual culture; the diameter of each data point indicates the product concentration achieved for that culture. [Figure 16C]Figures 16A–16D are a series of graphs showing bubble plots of specific productivity of cell cultures expressing the LMM gene and the gene of interest in aFOG. Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 16A), cergutuzumab (Figure 16B), infliximab (Figure 16C), or cB72.3 (Figure 16D). Cultures were grown in deep-well plates, and VCC was measured by Celigo cell count for each culture / well. Productivity was measured using an Octet instrument with a protein A biosensor. Each indicated data point corresponds to an individual culture; the diameter of each data point indicates the product concentration achieved for that culture. [Figure 16D] Figures 16A–16D are a series of graphs showing bubble plots of specific productivity of cell cultures expressing the LMM gene and the gene of interest in aFOG. Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 16A), cergutuzumab (Figure 16B), infliximab (Figure 16C), or cB72.3 (Figure 16D). Cultures were grown in deep-well plates, and VCC was measured by Celigo cell count for each culture / well. Productivity was measured using an Octet instrument with a protein A biosensor. Each indicated data point corresponds to an individual culture; the diameter of each data point indicates the product concentration achieved for that culture. [Figure 17A] Figures 17A–17D are a series of graphs showing 95% confidence interval plots of product concentrations of cell cultures expressing the LMM gene and the gene of interest in aFOG. Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 17A), cergutuzumab (Figure 17B), infliximab (Figure 17C), or cB72.3 (Figure 17D). Productivity was measured using Octet with a protein A biosensor. Each data point represents the mean productivity of the indicated culture with a confidence interval of p=0.95. [Figure 17B]Figures 17A–17D are a series of graphs showing 95% confidence interval plots of product concentrations of cell cultures expressing the LMM gene and the gene of interest in aFOG. Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 17A), cergutuzumab (Figure 17B), infliximab (Figure 17C), or cB72.3 (Figure 17D). Productivity was measured using Octet with a protein A biosensor. Each data point represents the mean productivity of the indicated culture with a confidence interval of p=0.95. [Figure 17C] Figures 17A–17D are a series of graphs showing 95% confidence interval plots of product concentrations of cell cultures expressing the LMM gene and the gene of interest in aFOG. Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 17A), cergutuzumab (Figure 17B), infliximab (Figure 17C), or cB72.3 (Figure 17D). Productivity was measured using Octet with a protein A biosensor. Each data point represents the mean productivity of the indicated culture with a confidence interval of p=0.95. [Figure 17D] Figures 17A–17D are a series of graphs showing 95% confidence interval plots of product concentrations of cell cultures expressing the LMM gene and the gene of interest in aFOG. Cells were transfected with the indicated LMM gene under the control of the indicated promoter, and genes encoding etanercept (Figure 17A), cergutuzumab (Figure 17B), infliximab (Figure 17C), or cB72.3 (Figure 17D). Productivity was measured using Octet with a protein A biosensor. Each data point represents the mean productivity of the indicated culture with a confidence interval of p=0.95. [Figure 18] Figure 18 shows a series of Western blots of cell solubilates from SCD1 or SREBF1-mediated CHO cells isolated using the P5CS metabolic selection system, and from cells grown in the absence of proline in the culture medium. [Figure 19A]Figures 19A–19D show a series of graphs of cell-specific productivity (Qp) to IVCs assessed in a miniature bioreactor under fed-batch conditions in the absence of glutamine or proline, from SREBF1 or SCD1-modified CHO cell pools that had varying levels of expression of these lipid metabolism alteration (LMM) genes, and were then transfected with genes for secretory expression of either etanercept or infliximab and recombinant protein molecules, along with the selective marker glutamine synthetase. [Figure 19B] Figures 19A–19D show a series of graphs of cell-specific productivity (Qp) to IVCs assessed in a miniature bioreactor under fed-batch conditions in the absence of glutamine or proline, from SREBF1 or SCD1-modified CHO cell pools that had varying levels of expression of these lipid metabolism alteration (LMM) genes, and were then transfected with genes for secretory expression of either etanercept or infliximab and recombinant protein molecules, along with the selective marker glutamine synthetase. [Figure 19C] Figures 19A–19D show a series of graphs of cell-specific productivity (Qp) to IVCs assessed in a miniature bioreactor under fed-batch conditions in the absence of glutamine or proline, from SREBF1 or SCD1-modified CHO cell pools that had varying levels of expression of these lipid metabolism alteration (LMM) genes, and were then transfected with genes for secretory expression of either etanercept or infliximab and recombinant protein molecules, along with the selective marker glutamine synthetase. [Figure 19D] Figures 19A–19D show a series of graphs of cell-specific productivity (Qp) to IVCs assessed in a miniature bioreactor under fed-batch conditions in the absence of glutamine or proline, from SREBF1 or SCD1-modified CHO cell pools that had varying levels of expression of these lipid metabolism alteration (LMM) genes, and were then transfected with genes for secretory expression of either etanercept or infliximab and recombinant protein molecules, along with the selective marker glutamine synthetase. [Modes for carrying out the invention]

[0008] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference as a whole. In addition, materials, methods and examples are illustrative and not intended to be limiting. Headings, subheadings or numbered or lettered elements, e.g., (a), (b), (i), etc., are provided solely for readability. The use of headings or numbered or lettered elements in this document does not require that the steps or elements be performed alphabetically or that the steps or elements be separated from each other. Other features, purposes and advantages of the present invention are evident from the description and drawings and the claims. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0009] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. In some embodiments, the article “a” or “an” refers to a single one of the grammatical objects of the article. In some embodiments, the article “a” or “an” refers to multiple (e.g., more than one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) grammatical objects of the article. For example, "a cell" can mean one cell or more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more).

[0010] The term “approximately” means that, when referring to measurable values ​​such as quantity or temporary duration, the variation includes such variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, when appropriate to do so in the manner disclosed.

[0011] As used herein, the term “plurality” refers to more than one of the grammatical objects of an article (e.g., two or more). For example, “plural cells” may mean two or more cells.

[0012] As used herein, the term “endogenous” refers to any material from an organism, cell, tissue, or system, or any material naturally produced within them.

[0013] As used herein, the term “heterogeneous” refers to any material introduced into an organism, cell, tissue, or system, and / or any material produced outside of them. Thus, “heterogeneous nucleic acid” refers to nucleic acid introduced into an organism, cell, tissue, or system, and / or nucleic acid produced outside of them. In some embodiments, the sequence of the heterogeneous nucleic acid is not produced or found naturally inside the organism, cell, tissue, or system into which the heterogeneous nucleic acid is introduced. In some embodiments, the sequence of the heterogeneous nucleic acid is found naturally inside the organism, cell, tissue, or system (e.g., nucleic acid that is found naturally in cells and encodes, for example, an enzyme or LMM as described herein (wherein the nucleic acid is then expressed in the cell to produce additional copies of the enzyme or LMM)). Similarly, “heterogeneous polypeptide” refers to a polypeptide introduced into an organism, cell, tissue, or system, and / or polypeptide produced outside of them. In some embodiments, the polypeptide is not produced or found naturally inside the organism, cell, tissue, or system into which the heterogeneous polypeptide is introduced, for example, by expression from a heterogeneous nucleic acid sequence. In some embodiments, heterologous polypeptides can be found naturally within an organism, cell, tissue, or system (e.g., ectopically expressed in cells, naturally occurring in cells, such as enzymes or LMMs described herein). In certain embodiments, heterologous nucleic acids or polypeptides are introduced into an organism, cell, tissue, or system containing an endogenous copy of the same polypeptide or nucleic acid, thereby increasing the amount of polypeptide or nucleic acid in the organism, cell, tissue, or system. In certain cases, the term “heterologous” may refer to any material from one species when introduced into an organism, cell, tissue, or system from a different species. In some cases, the terms “heterologous” and “exogenous” are used interchangeably.

[0014] As used herein, the term “enzyme molecule” refers to a polypeptide having the desired enzymatic activity. An enzyme molecule may share structural similarities (e.g., sequence homology) with an enzyme having the desired enzymatic activity. In some cases, an enzyme molecule has at least 50% amino acid sequence identity to an enzyme having the desired enzymatic activity (e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In some cases, when used as an identifier for an enzyme (e.g., pyrroline-5-carboxylate synthase (P5CS)), the term “molecule” refers to a polypeptide having the enzymatic activity of the specified enzyme. For example, the term “P5CS molecule” as used herein refers to a polypeptide having the enzymatic activity of P5CS. In some cases, the P5CS molecule has at least 50% amino acid sequence identity to the P5CS enzyme (e.g., mammalian P5CS) (e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0015] As used herein, the terms “nucleic acid,” “polynucleotide,” or “nucleic acid molecule” are interchangeable and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations thereof, in either single-stranded or double-stranded form, or polymers thereof. The term “nucleic acid” includes, but is not limited to, genes, cDNA, or RNA sequences (e.g., mRNA). In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized or artificial) or recombinant. Unless otherwise specified, the term includes molecules containing analogs or derivatives of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to natural or non-natural nucleotides. Unless otherwise indicated, and a particular nucleic acid sequence implicitly includes not only the explicitly indicated sequence, but also its conserved variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences. In particular, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605~2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91~98 (1994)). As used herein, “target nucleic acid” means any nucleic acid of interest that can be desirablely introduced into a cell as described herein, or may be present in a cell, and includes, for example, a sequence encoding the product described herein.

[0016] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds consisting of amino acid residues covalently linked by peptide bonds or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may constitute a protein sequence or peptide sequence. In one embodiment, a protein may consist of more than one polypeptide, e.g., two, three, four, five, or more, each polypeptide associated with another polypeptide by either covalent or non-covalent bonding / interaction. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds or by means other than peptide bonds. As used herein, this term refers to both short chains, commonly called, for example, peptides, oligopeptides, and oligomers in the art, and long chains, commonly called proteins in the art, of which there are many types. Among the many types of polypeptides, there are, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins.

[0017] When the term is used herein, “product” means, for example, a molecule, for example, expressed by a cell, for example, a cell modified or manipulated to produce a product, such as a polypeptide, for example, a protein, for example, a glycoprotein, nucleic acid, lipid, sugar, polysaccharide, or any hybrid thereof. In one embodiment, the product is a protein or polypeptide product. In one embodiment, the product includes a natural product. In one embodiment, the product includes a non-natural product. In one embodiment, part of the product is natural, while another part of the product is non-natural. In one embodiment, the product is a polypeptide, for example, a recombinant polypeptide. In one embodiment, the product is suitable for diagnostic or preclinical use. In another embodiment, the product is suitable for therapeutic use, for example, for the treatment of a disease. In some embodiments, the product is a protein product. In some embodiments, the product is a recombinant protein or therapeutic protein, as described herein, for example, in Tables 5-8.

[0018] As used herein, the term “promoter” refers to a sequence having sufficient sequence, for example, from a natural or engineered promoter, such that operably linking a coding sequence to the promoter results in the expression of the coding sequence. For example, a cytomegalovirus (CMV) promoter includes all or an active fragment of a CMV promoter, for example, all or an active fragment of a CMV promoter that optionally includes an intron A and / or UTR sequence. In one embodiment, a CMV promoter differs from a natural or engineered variant CMV promoter by 5, 10, 20, 30, 50, or 100 or fewer nucleotides. In one embodiment, a CMV promoter differs from a natural or engineered variant CMV promoter by 1, 5, 10, or 50% or fewer of its nucleotides. As used herein, a promoter may be a constitutive, regulated, repressive, strong, weak, or other property of the promoter sequence it contains. In one embodiment, a promoter may include a coding sequence, for example, the 5' or 3' sequence of a coding sequence of a recombinant polypeptide, therapeutic polypeptide, or repressor polypeptide. In one embodiment, the promoter may include a sequence within one or more introns of a gene, for example, a recombinant polypeptide, a therapeutic polypeptide, or a repressor polypeptide. In one embodiment, the promoter may be included in part or as a whole within the 5' or 3' sequence of a coding sequence, for example, a recombinant polypeptide, a therapeutic polypeptide, or a repressor polypeptide. In one embodiment, the promoter may be included in part or as a whole within a coding sequence, for example, a recombinant polypeptide, a therapeutic polypeptide, or a repressor polypeptide. In one embodiment, the promoter may be included in part or as a whole within one or more introns of a gene, for example, a recombinant polypeptide, a therapeutic polypeptide, or a repressor polypeptide.

[0019] As used herein, the term “operably linked” refers to a relationship between a nucleic acid sequence encoding a polypeptide and a control element in which the polypeptide-encoding sequence and the control element are operably linked, such that the control element is arranged in a manner suitable for regulating the expression of the product-encoding sequence (e.g., polypeptide). Thus, for various control elements, the operably linked sequences constitute various arrangements of the product-encoding sequence with respect to the control element. For example, if the promoter element and the product-encoding sequence are located proximal to each other and on the same nucleic acid, the product-encoding sequence may be operably linked to a control element containing the promoter element. In another example, if the enhancer sequence and the product-encoding sequence are located a suitable number of bases apart on the same nucleic acid, or even on separate and distinct nucleic acids, the product-encoding sequence may be operably linked to a control element containing a distally acting enhancer sequence.

[0020] As used herein, the term “regulatory element” refers to a coding sequence, such as a nucleic acid suitable for regulating (e.g., increasing or decreasing) the expression of a gene. A regulatory element may include a promoter sequence, an enhancer sequence, or both a promoter sequence and an enhancer sequence. A regulatory element may include a continuous nucleic acid sequence, a discontinuous nucleic acid sequence (a sequence interrupted by another coding or non-coding nucleic acid sequence), or both. A single regulatory element may be contained in a single nucleic acid or more than one nucleic acid. In one embodiment, a regulatory element may include the 5' or 3' sequence of a coding sequence, such as a recombinant polypeptide, a therapeutic polypeptide, or a repressor polypeptide. In one embodiment, a regulatory element may include a sequence within one or more introns of a gene, such as a gene encoding a recombinant polypeptide, a therapeutic polypeptide, or a repressor polypeptide. In one embodiment, a regulatory element may be contained in part or as a whole within the 5' or 3' sequence of a coding sequence, such as a recombinant polypeptide, a therapeutic polypeptide, or a repressor polypeptide. In one embodiment, the regulatory element may be contained in part or as a whole within a coding sequence, such as a recombinant polypeptide, therapeutic polypeptide, or repressor polypeptide. In one embodiment, the regulatory element may be contained in part or as a whole within one or more introns of a gene, such as a gene encoding a recombinant polypeptide, therapeutic polypeptide, or repressor polypeptide. In one embodiment, a single regulatory element may include i) a nucleic acid sequence proximal to a gene, such as a gene encoding a recombinant polypeptide, therapeutic polypeptide, or repressor polypeptide (e.g., adjacent to or contained within the gene), or ii) a nucleic acid sequence distal to a gene, such as a gene encoding a recombinant polypeptide, therapeutic polypeptide, or repressor polypeptide (e.g., separated by 10 or more, 100 or more, 1,000 or more, or 10,000 or more bases, or located on separate or distinct nucleic acids).

[0021] When used herein, "lipid metabolism modifier" or "LMM" means: the expression of components involved in the lipid metabolic pathway (e.g., transcription or translation); the activity of components involved in the lipid metabolic pathway, e.g., gene products (e.g., enzyme activity); the level or amount of lipids present in the cell; the level or amount of lipid rafts or the rate of lipid raft formation; the fluidity, permeability, or thickness of the cell membrane, e.g., the plasma membrane or organelle membrane; the conversion of saturated lipids to unsaturated lipids or vice versa; the level or amount of saturated or unsaturated lipids in the cell, e.g., monounsaturated lipids; the lipid composition to achieve a preferred lipid composition that has a favorable effect on ER activity; ER dilation; Golgi apparatus dilation; secretory vesicles or the level or amount of secretory vesicle formation; the level or rate of secretion; membrane receptors (e.g., ATR) (e.g., The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR. Zhang XH, Zhao C, Ma ZA. J Cell Sci.) See also ATR (ataxia telangiectasia mutated- and Rad3-related kinase) is activated by mild hypothermia in mammalian cells and subsequently activates p53. Roobol A, Roobol J, Carden MJ, Bastide A, Willis AE, Dunn WB, Goodacre R, Smales CM. Biochem J. April 15, 2011;435(2):499~508. doi:10.1042 / BJ20101303. PMID:21284603) and SREPB (e.g., Int J Biol Sci. March 21, 2016;12(5):569~79). doi:10.7150 / ijbs.14027. eCollection 2016.This refers to the activation or inactivation of additional receptors (see, for example, Biochim Biophys Acta. March 17, 2016 pii:S1388~1981(16)30071~3. doi:10.1016 / j.bbalip.2016.03.019), and / or molecules, gene products, polypeptides, or enzymes that modify, for example, increase or decrease one or more of the unfolded protein response (UPR). In one embodiment, LMM comprises a polypeptide. In one embodiment, LMM comprises a transcription regulator, for example, a transcription factor. In one embodiment, LMM comprises SREBF1 or a functional fragment thereof (for example, SREBF-410). In one embodiment, LMM comprises an enzyme. In another embodiment, LMM comprises SCD1 or a functional fragment thereof.

[0022] Methods for identifying, selecting, or culturing cells In one embodiment, the invention of the present disclosure relates to a method for identifying, selecting and / or culturing cells. In some embodiments, the method is a)(i) Target nucleic acid sequence and (ii) If expressed, nucleic acid sequences that result in high levels of activity of enzymes in the amino acid synthesis pathway, for example, nucleic acid sequences that encode enzyme molecules containing activity A step of preparing cells containing nucleic acids, such as vectors, such as replicable vectors or integration vectors, b) A step of culturing cells containing nucleic acid sequences under conditions sufficient to allow the growth of cells containing nucleic acid sequences, in the presence of a medium having insufficient levels of amino acids to support the growth of cells that are the same as cells that do not have high activity. This includes, thereby enabling the identification, selection, or culture of cells containing heterogeneous nucleic acid sequences.

[0023] In some embodiments, the enzyme molecule catalyzes the rate-limiting step in the amino acid biosynthesis pathway. In other embodiments, the enzyme molecule catalyzes a rate-non-rate-limiting step in the amino acid biosynthesis pathway. In these embodiments, the amino acid is proline, tyrosine, or tryptophan.

[0024] In some embodiments, the cells further include an inhibitor of the activity of the enzyme molecule. The inhibitor may be used to increase the rigor of the selection process by reducing or preventing endogenous enzyme molecule activity, for example, so that cells that do not take up nucleic acids containing the target nucleic acid sequence exhibit low or undetectable levels of endogenous enzyme molecule activity. Cells exhibiting low or undetectable levels of endogenous enzyme molecule activity may not be able to grow and / or survive in the absence of an external supply of amino acids (e.g., proline, tyrosine, or tryptophan) whose synthesis requires the activity of the enzyme molecule. In some embodiments, the inhibitor binds to the enzyme molecule, for example, and inhibits it. In embodiments, the inhibitor is an allosteric inhibitor of the enzyme molecule. In embodiments, the inhibitor is a competitive inhibitor of the enzyme molecule. In some embodiments, the inhibitor inhibits the transcription or translation of the enzyme molecule, for example, endogenous transcription or translation of the enzyme molecule. In some embodiments, the inhibitor inhibits enzyme molecules in the biosynthetic pathway of proline, tyrosine, or tryptophan. In the embodiment, the inhibitor inhibits the activity of the pyrroline-5-carboxylic acid synthase (P5CS) molecule. In the embodiment, the inhibitor inhibits the activity of P5CS. In the embodiment, the inhibitor is a proline analog. In the embodiment, the inhibitor is L-azetidine-2-carboxylic acid, 3,4-dehydro-L-proline, or L-4-thiazolidinedcarboxylic acid.

[0025] In some embodiments, the method further includes one or more additional culture steps. In some embodiments, one or more additional culture steps include culturing cells in the presence of a second medium (e.g., the same medium composition as used in the first culture step, or a different medium than the one used in the first culture step). In embodiments, the first culture step utilizes a medium containing an insufficient level of a first amino acid (e.g., proline), for example, a medium lacking the first amino acid, and the second culture step utilizes a medium containing an insufficient level of a second amino acid (e.g., tyrosine or tryptophan), for example, a medium lacking the second amino acid. In embodiments, only if the cells further contain the target nucleic acid of interest, the cells include an enzyme molecule that rescues the production of the first amino acid and / or an enzyme molecule that rescues the production of the second amino acid. This method can be extended to additional culture steps, each culture step intended to include a medium containing insufficient levels of amino acids (e.g., the same or different amino acids not present in the first or second culture medium), such as an amino acid-deficient medium, and cells containing enzyme molecules that can rescue the production of amino acids (e.g., the same or different amino acids not present in the first or second culture medium). In embodiments, the medium used in the additional culture steps includes an inhibitor of amino acids not present in the culture used in the additional culture steps. In some embodiments, the culture steps may be carried out simultaneously using a medium containing, for example, several amino acids in insufficient levels, such as a medium lacking several amino acids. In some embodiments, only one subset of culture steps includes the use of an enzyme molecule inhibitor. For example, a first culture step may include the use of an enzyme molecule inhibitor, followed by culture steps that do not include the use of an enzyme molecule inhibitor.

[0026] In some embodiments, this method can be used to generate cells containing heterogeneous nucleic acids, for example, products, for example, polypeptide products, for example, cells useful for producing antibodies.

[0027] Enzyme molecules and their inhibitors The present invention generally features methods and compositions for selecting cells into which nucleic acids have been introduced by selectively expressing enzyme molecules involved in amino acid biosynthesis. Enzyme molecules can be introduced into cells along with the target nucleic acid. For example, a nucleic acid containing both the target nucleic acid and the gene encoding the enzyme molecule can be introduced into cells so that the enzyme molecule is expressed within the cell. In some embodiments, cells containing the target nucleic acid exhibit higher enzyme molecule activity compared to cells lacking the target nucleic acid. In some embodiments, the level of enzyme molecule activity increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, or more compared to the detectable enzyme molecule activity in cells lacking the target nucleic acid. In some embodiments, cells with high activity can grow more rapidly than cells lacking the target nucleic acid. In some embodiments, the cells contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 5000, 10,000 copies or more of nucleic acid encoding the enzyme. In embodiments, the highly active cells grow at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 5000, or 10,000 times faster in an amino acid-deficient medium than in cells lacking the nucleic acid of interest. Subsequently, the cells can be grown in an amino acid-deficient medium to select cells that have taken up the target nucleic acid. In some embodiments, selection involves selecting one or more cells that exhibit growth in the amino acid-deficient medium.

[0028] Enzyme molecule inhibitors The cells described herein may, in some embodiments, further include inhibitors of enzyme molecules expressed by nucleic acids (e.g., the target nucleic acid or a second nucleic acid) introduced into the cells. The inhibitors may act, for example, to reduce or block the activity of enzyme molecules endogenously produced by the cells and / or to reduce or block the growth of revertant cells, thereby increasing the rigor of the selection methods described herein. In some embodiments, the intracellular level of the inhibitor is sufficient to reduce the endogenous enzyme molecule activity to less than about 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 40%, or 50% of the activity observed in cells lacking the inhibitor. In some embodiments, less than about 0.001%, 0.01%, 0.1%, 1%, 5%, or 10% of the cells selected based on growth in an amino acid-deficient medium do not contain the target nucleic acid. In some embodiments, the ratio of enzyme molecules to inhibitor molecules within the cell is approximately 1:1000, 1:500, 1:250, 1:200, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 250:1, 500:1, or 1000:1.

[0029] The inhibitor may be, for example, an amino acid or its analogue, a polypeptide, a nucleic acid, or a small molecule. In some embodiments, the inhibitor is an analogue of an amino acid produced by a biosynthetic pathway in which an enzyme molecule participates. In some embodiments, the inhibitor is an antibody molecule (e.g., an antibody or antibody fragment, as described herein), a fusion protein, a hormone, a cytokine, a growth factor, an enzyme, a glycoprotein, a lipoprotein, a reporter protein, a therapeutic peptide, an aptamer, or a structural and / or functional fragment or hybrid of any of these. In some embodiments, the inhibitor is antisense RNA, siRNA, tRNA, ribosomal RNA, microRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, or a long non-coding RNA.

[0030] Non-limiting examples of enzyme molecules and their inhibitors that may be used in the compositions and methods described herein include the enzyme molecules and their inhibitors listed in Table 1.

[0031] [Table 1] TIFF2026048662000002.tif190149 TIFF2026048662000003.tif200149 TIFF2026048662000004.tif212149 TIFF2026048662000005.tif208149 TIFF2026048662000006.tif212149 TIFF2026048662000007.tif209149 TIFF2026048662000008.tif204149 TIFF2026048662000009.tif207149 TIFF2026048662000010.tif138149

[0032] In some embodiments, the inhibitor inhibits enzyme molecules in the biosynthetic pathway of amino acids, such as proline, tyrosine, or tryptophan. In some embodiments, the inhibitor inhibits enzyme molecules that catalyze the rate-limiting step in the amino acid biosynthesis pathway.

[0033] In some embodiments, the inhibitor inhibits an enzyme molecule in the proline biosynthesis pathway, such as an enzyme molecule that catalyzes the rate-limiting step in the proline biosynthesis pathway. In embodiments, the inhibitor inhibits the activity of the pyrroline-5-carboxylic acid synthase (P5CS) molecule. In embodiments, the inhibitor inhibits the activity of P5CS. In embodiments, the inhibitor is proline or a proline analog. In embodiments, the inhibitor is L-azetidine-2-carboxylic acid, 3,4-dehydro-L-proline, or L-4-thiazolidinedcarboxylic acid.

[0034] In some embodiments, the inhibitor inhibits an enzyme molecule in the tyrosine biosynthesis pathway, such as an enzyme molecule that catalyzes the rate-limiting step in the tyrosine biosynthesis pathway. In embodiments, the inhibitor is a tyrosine analog.

[0035] In some embodiments, the inhibitor inhibits an enzyme molecule in the tryptophan biosynthesis pathway, such as an enzyme molecule that catalyzes the rate-limiting step in the tryptophan biosynthesis pathway. In embodiments, the inhibitor is a tryptophan analog.

[0036] Lipid metabolism altering factors This disclosure features methods and compositions for producing cells containing heterogeneous nucleic acids. In some cases, the cells further include lipid metabolism modifiers (LMMs), generally under the control of promoters, which can alter lipid metabolism within the cell. In embodiments, the LMMs include inclusive regulators that affect numerous aspects of pathways or processes involved in lipid metabolism, such as denomination of lipids, fatty acid re-esterification, fatty acid saturation or desaturation, fatty acid elongation, and phospholipid biosynthesis pathways. For example, the inclusive regulator is located upstream of one or more lipid metabolic pathways or processes, such that the inclusive regulator affects several, e.g., two or more, downstream processes or components of lipid metabolism. In one embodiment, the inclusive regulator is a transcription factor that can activate the expression of one or more, e.g., two or more, target genes involved in different lipid metabolic processes or pathways. Thus, without being bound by any theory, the use of inclusive regulators described herein may result in a greater increase in the productivity, robustness, and survival of cells compared to the use of downstream effectors that modify only a single target or other component of lipid metabolism. While not bound by any particular theory, comprehensive or broader alterations of numerous lipid metabolic pathways are thought to increase cellular productivity by affecting more processes involved in improving cellular productivity, product quality, and robustness.

[0037] Lipid metabolic pathways as described herein refer to processes relating to the synthesis, degradation, transformation, or modification of lipids or lipid-related molecules. Lipid molecules include, but are not limited to, fatty acids, glycerolipids, glycerophospholipids, phospholipids, saccharolipids, sphingolipids, and sterol lipids, such as cholesterol, and polyketides. Examples of lipid metabolic pathways include, but are not limited to, novel lipid synthesis, fatty acid re-esterification, fatty acid saturation, fatty acid desaturation, fatty acid elongation, and phospholipid biosynthesis. In one embodiment, the method described herein provides cells comprising a modification that alters lipid metabolism. A modification that alters lipid metabolism may be an agent that increases or decreases the expression of components involved in lipid metabolism. In one embodiment, the modification that alters lipid metabolism comprises an exogenous nucleic acid encoding a lipid metabolism modifier (LMM). In such embodiments, the exogenous nucleic acid encoding the LMM is introduced into cells by one of the nucleic acid delivery methods or techniques described herein, such as transduction or transfection.

[0038] In some embodiments, the methods described herein provide cells comprising one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications that alter lipid metabolism. In embodiments in which cells comprise two or more modifications that alter lipid metabolism, each modification that alters lipid metabolism comprises an exogenous nucleic acid encoding a lipid metabolism molecule (LMM). In one embodiment, each of the two or more exogenous nucleic acids encoding the LMM may be located within the same nucleic acid molecule or within two or more different nucleic acid molecules. In such embodiments in which cells comprise two or more nucleic acid sequences encoding the LMM, the LMMs are distinct from one another, for example, encoding different polypeptide sequences or having different functions. In some embodiments, multiple different heterologous LMMs (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more, e.g., LMMs described herein) are expressed in the cell. In certain embodiments, one or more of the heterologous LMMs are spontaneously expressed by the cell. In certain embodiments, one or more of the heterologous LMMs are not spontaneously expressed by the cell.

[0039] In one embodiment, a modification altering lipid metabolism increases or decreases the expression or activity of components involved in one or more lipid metabolic pathways. In some embodiments, a modification of cellular lipid metabolism results in a change in the spatial distribution of lipids within the cell, e.g., within the cell membrane (e.g., within lipid rafts), organelles (e.g., endoplasmic reticulum, Golgi apparatus, nucleus, lysosomes, peroxisomes, vacuoles and / or mitochondria), exosomes, droplets, or any other region or compartment of the cell containing lipids. In embodiments where a modification altering lipid metabolism results in increased expression, e.g., transcription or translation, or increased activity of components in a lipid metabolic pathway, the component is a positive regulator of the lipid metabolic pathway. In embodiments where a modification altering lipid metabolism results in decreased expression, e.g., decreased transcription, translation, turnover and / or degradation, or decreased activity of components in a lipid metabolic pathway, the component is a negative regulator of the lipid metabolic pathway. Assays for quantifying the expression of genes in lipid metabolic pathways, such as transcription and / or translation, are known in the art and include quantification of the amount of mRNA encoding a gene; or quantification of the amount of a gene product or polypeptide; PCR-based assays, such as quantitative real-time PCR; Northern blotting; or microarrays. Assays for quantifying the activity of components of lipid metabolic pathways, such as enzymes in lipid metabolic pathways, are specific to particular components of lipid metabolic pathways.

[0040] In embodiments where altering cellular lipid metabolism results in an increase in intracellular lipid levels or quantities, the total intracellular lipid levels or quantities may be increased. In some embodiments, altering cellular lipid metabolism results in altering the spatial distribution of intracellular lipids, e.g., within the cell membrane (e.g., within lipid rafts), organelles (e.g., endoplasmic reticulum, Golgi apparatus, nucleus, lysosomes, peroxisomes, vacuoles and / or mitochondria), exosomes, droplets, or any other region or compartment of the cell containing lipids. In one embodiment, altering lipid metabolism results in increased cell viability. In one embodiment, altering lipid metabolism results in increased culture viability. In one embodiment, altering lipid metabolism results in increased cell proliferation. In one embodiment, altering lipid metabolism results in increased productivity, e.g., increased amount, quantity, or yield of the product produced, or increased production rate. In one embodiment, modifications that alter lipid metabolism result in increased product quality, such as aggregation, glycosylation state or heterogeneity, fragmentation, proper folding or assembly, post-translational modification, or disulfide bond scrambling.

[0041] In one embodiment, LMM is overexpressed intracellularly by increasing its expression, for example, by introducing a heterologous nucleic acid encoding LMM (e.g., a nucleic acid derived from outside the cell, such as a nucleic acid construct containing the gene encoding LMM), or by introducing a promoter element or other regulatory transcription element. In some embodiments, the cell contains an endogenous copy of LMM that has been overexpressed intracellularly. In other embodiments, the cell does not contain an endogenous copy of LMM that has been overexpressed intracellularly. In yet another embodiment, LMM expression or activity is inhibited or reduced, for example, by introducing an LMM inhibitor or exogenous inhibitory nucleic acid, such as an RNA interference agent. Examples of inhibitory nucleic acids include short interfering RNA (siRNA) and short hairpin RNA (shRNA), such as mRNA encoding LMM, that target LMM. In one embodiment, LMM activity or expression is increased or decreased by post-translational modification or by altering other endogenous regulatory mechanisms that regulate LMM activity or expression. Post-translational modification (PMI) regulation includes, but is not limited to, phosphorylation, SUMOylation, ubiquitination, acetylation, methylation, or glycosylation, which can increase or decrease LMM expression or activity. For example, PMI regulation may be achieved through alteration of enzymes or molecules that modify LMM, or through modifications of LMM, such that PMI occurs more frequently or constitutively. PMI regulation may also include altering endogenous regulatory mechanisms, such as increasing or decreasing one or more of the following, which can increase or decrease LMM expression or activity: miRNA regulation, protein cleavage, expression of specific isoforms, alternative splicing, and degradation. In one embodiment, LMM alters, for example, the expression, e.g., transcription, or activity of components of lipid metabolic pathways, e.g., increasing or decreasing. In another embodiment, LMM alters, for example, the synthesis, degradation, elongation, or structural conformation (e.g., saturation, desaturation, or esterification) of lipids or lipid-related molecules, e.g., increasing or decreasing. Exemplary components of LMM and / or lipid metabolic pathways are listed below, but are not limited to those listed in Table 2.

[0042] [Table 2] TIFF2026048662000012.tif193149 TIFF2026048662000013.tif49149

[0043] In embodiments, the LMM contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity or homology with components involved in lipid metabolic pathways, such as gene products, provided, for example, in Table 2; or differs from the amino acid sequence of components involved in lipid metabolic pathways, such as gene products, provided, for example, in Table 2, by 1, 2, 3, 20, 15, or 10 or fewer amino acid residues.

[0044] In embodiments, the LMM comprises a functional fragment of a component involved in the lipid metabolic pathway, as provided, for example, in Table 2. The functional fragments of the LMM described herein may comprise one or more functional domains of the LMM. For example, a functional fragment of an LMM that is a transcription factor comprises a DNA-binding domain and a transactivation domain. For example, a functional fragment of an LMM that is an enzyme comprises a domain having enzymatic activity. The functional fragments of the LMM described herein retain the functional activity of the full-length LMM, for example, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the functional activity. The functional fragments of the LMM may be determined experimentally by those skilled in the art, or they may be predicted using an algorithm based on the sequence homology of the functional domains. Exemplary LMMs are further described below.

[0045] In any embodiment of the methods described herein, LMM may be a transcription regulator. In embodiments, LMM is a transcription factor or transcription activator that binds to DNA or associates in a complex that binds to DNA and recruits or associates in a complex that recruits RNA polymerase for the transcription of one or more gene products involved in lipid metabolism. In embodiments, LMM binds to a sterol-binding element and / or an E-box promoter sequence. In embodiments, LMM comprises sterol regulatory element binding factor 1 (SREBF1) or sterol regulatory element binding factor 2 (SREBF2) or a functional fragment or isoform thereof.

[0046] In some embodiments, the LMM includes a comprehensive transcription activator or transcription factor. In embodiments, the LMM can alter the transcription of two or more components of lipid metabolic pathways, e.g., 2, 3, 4, 5, 6, or more, as provided, for example, in either Table 3 or 4. In embodiments, the LMM can alter the transcription of one or more components of two or more lipid metabolic pathways, e.g., 1, 2, 3, 4, or 5, or more, e.g., components and pathways provided, e.g., in Table 2.

[0047] Sterol regulatory element binding factor 1 (SREBF1) is a comprehensive transcriptional activator that upregulates the transcription of genes involved in lipid synthesis, fatty acid reesterification, fatty acid desaturation and elongation, and phospholipid biosynthesis by binding to sterol regulatory elements (SREs) and E-box promoter sequences (Hagen, Rodriguez-Cuenca et al. 2010) present in the promoter regions of target genes. The transcription of the SREBF1 gene itself is intrinsically regulated by the presence of sterol regulatory elements (SREs) among other transcriptional regulatory elements in the gene's promoter region. Furthermore, numerous posttranslational regulatory mechanisms, including phosphorylation, ubiquitination, SUMOylation, acetylation, fatty acid-mediated modification, and proteolytic processing, are anchored around SREBF1, contributing to a rigidly regulated yet adaptable homeostatic system.

[0048] In embodiments, LMM includes an enzyme. In embodiments, LMM includes an enzyme that converts saturated fatty acids to unsaturated fatty acids. In embodiments, LMM includes an enzyme that converts saturated fatty acids to monounsaturated fatty acids, such as fatty acids having one double bond. In embodiments, LMM includes an enzyme that converts saturated fatty acids to polyunsaturated fatty acids, such as fatty acids having more than one, for example, two, three, four, five or more double bonds. In embodiments, LMM includes stearoyl CoA desaturase 1 (SCD1), stearoyl CoA desaturase 2 (SCD2), stearoyl CoA desaturase 3 (SCD3), stearoyl CoA desaturase 4 (SCD4), stearoyl CoA desaturase 5 (SCD5), its isoforms or functional fragments thereof.

[0049] In some embodiments, the LMM present in the cells described herein is under the control of a promoter. The promoter is preferably fitted to the LMM gene to maximize LMM production by the cell. Non-limiting examples of promoters that may be used to regulate LMM expression include the SV40, mCMV, and PGK promoters. Non-limiting examples of promoter-LMM gene combinations that may be used, for example, in any of the methods or compositions (e.g., cells) described herein are shown in Table 3. Each of these combinations is intended to be present in cells containing P5CS when introduced into cells, for example, according to the methods described herein.

[0050] [Table 3]

[0051] Any of the promoter-LMM combinations described herein may exist in further combination with a gene encoding an enzyme molecule, for example, in any of the cells, nucleic acids, or other compositions described herein. For example, any of the promoter-LMM combinations described herein may exist in a cell further comprising a nucleic acid encoding a P5CS molecule and containing the target nucleic acid described herein. Furthermore, any of the promoter-LMM combinations described herein may exist in a cell having an inhibitor of an amino acid biosynthesis enzyme molecule (for example, the enzyme molecule is also present in the cell). For example, any of the promoter-LMM combinations described herein may exist in a cell further comprising (i) a nucleic acid encoding a P5CS molecule and containing the target nucleic acid described herein, and (ii) a P5CS inhibitor, for example, L-azetidine-2-carboxylic acid, 3,4-dehydro-L-proline, or L-4-thiazolidinedic acid. For example, non-limiting examples of promoter-LMM-P5CS inhibitor combinations that may be used in any of the methods or compositions (e.g., cells) described herein are shown in Table 4. Each of these combinations is intended to be present in cells containing P5CS when introduced into cells, for example, according to the methods described herein.

[0052] [Table 4] TIFF2026048662000016.tif182149

[0053] Cells and cell cultures In one aspect, the disclosure relates to methods for evaluating, classifying, identifying, selecting, or producing cells or cell lines that produce products, such as recombinant polypeptides or therapeutic polypeptides or nucleic acid molecules as described herein. In another aspect, the disclosure relates to methods and compositions for evaluating, classifying, identifying, selecting, or producing cells or cell lines having improved productivity and product quality, such as increased productivity. Generally, the methods herein can be used to produce cells or cell lines containing a nucleic acid construct (e.g., a genome-integrated vector or heterologous nucleic acid) comprising (i) a target nucleic acid sequence encoding the product of interest and (ii) a nucleic acid sequence encoding an enzyme molecule participating in the amino acid biosynthesis pathway, wherein the cells or cell lines do not endogenously express the enzyme molecule.

[0054] In one embodiment, the cells are mammalian cells. In another embodiment, the cells are non-mammalian cells. In one embodiment, the cells are from a mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, dog, horse, ferret, or cat. In another embodiment, the cells are mammalian cells, such as human cells, or rodent cells, such as hamster cells, mouse cells, or rat cells. In yet another embodiment, the cells are from a duck, parrot, fish, insect, plant, fungus, or yeast.

[0055] In one embodiment, the cells are Chinese hamster ovary (CHO) cells. In one embodiment, the cells are CHO-K1 cells, CHOK1SV cells, DG44 CHO cells, DUXB11 CHO cells, CHO-S, CHO GS knockout cells, CHOK1SV FUT8 knockout cells, CHOZN, or CHO-derived cells. CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1SV GS knockout cells (Lonza Biologics, Inc.). CHO FUT8 knockout cells are, for example, Potelligent® CHOK1SV FUT8 knockout (Lonza Biologics, Inc.).

[0056] In the embodiment, the cells are HeLa, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK (baby hamster kidney cells), VERO, SP2 / 0, NS0, YB2 / 0, Y0, EB66, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, CHOK1, CHOK1SV, Potelligent® (CHOK1SV FUT8-KO), CHO GS knockout, Xceed® (CHOK1SV GS-KO), CHOS, CHO DG44, CHO DXB11, and CHOZN, or any cells derived therefrom.

[0057] In this embodiment, eukaryotic cells are stem cells. Stem cells may include, for example, embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs).

[0058] In one embodiment, the cells are differentiated forms of any of the cells described herein. In one embodiment, the cells are cells derived from any primary cells in a culture.

[0059] In the embodiments, the cells are hepatocytes such as human hepatocytes, animal hepatocytes, or non-parenchymal cells. For example, the cells may be seedable metabolic human hepatocytes, seedable induction human hepatocytes, seedable Qualyst Transporter Certified (Trademark) human hepatocytes, human hepatocytes for suspension (including pooled hepatocytes from 10 and 20 donors), human hepatic Kupffer cells, human hepatic stellate cells, canine hepatocytes (including single and pooled beagle hepatocytes), mouse hepatocytes (including CD-1 and C57Bl / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including cynomolgus or rhesus monkey hepatocytes), feline hepatocytes (including domestic cat shorthair hepatocytes), and rabbit hepatocytes (including New Zealand white hepatocytes). Exemplary liver cells are commercially available from Triangle Research Labs, LLC, 6 Davis Drive, Research Triangle Park, North Carolina, USA 27709.

[0060] In some embodiments, the cells contain a knockout of glutamine synthase (GS). In embodiments, the cells do not contain a functional GS gene. In embodiments, the cells do not contain a GS gene. In embodiments, the intracellular GS gene contains a mutation that prevents the gene from encoding a functional GS protein.

[0061] In the embodiment, eukaryotic cells include, for example, yeast cells (e.g., Pichia genus (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta)), Komagataella genus (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii)), Saccharomyces genus (e.g., budding yeast (Saccharomyces cerevisiae), budding yeast, Saccharomyces kluyveri, Saccharomyces ubalaam) These are lower eukaryotic cells such as *Pichia pastris*, *Kluyveromyces* (e.g., *Kluyveromyces lactis*, *Kluyveromyces marxianus*), *Candida* (e.g., *Candida utilis*, *Candida cacaoi*, *Candida boidinii*), *Geotrichum* (e.g., *Geotrichum fermentans*), *Hansenula polymorpha*, *Yarrowia lipolytica*, or fission yeast (*Schizosaccharomyces pombe*). *Pichia pastris* species are preferred. Examples of Pichia pastris varieties include X33, GS115, KM71, KM71H, and CBS7435.

[0062] In the embodiment, eukaryotic cells are fungal cells (e.g., Aspergillus sp. (Aspergillus niger, Aspergillus fumigatus, Aspergillus orzyae, Aspergillus nidulans, etc.), Acremonium sp. (Acremonium thermophilum, etc.), Chaetomium sp. (Chaetomium thermophilum, etc.), Chrysosporium sp. (Chrysosporium thermophilum, etc.), Cordyceps sp. (Cordyceps militaris, etc.), Corynascus sp. * (sp.) , Ctenomyces sp., Fusarium sp. (e.g., Fusarium oxysporum), Glomerella sp. (e.g., Glomerella graminicola), Hypocrea sp. (e.g., Hypocrea jecorina), Magnaporthe sp. (e.g., M. orzyae), Myceliophthora sp. (e.g., Myceliophthora thermophile), Nectria sp. (e.g., Nectria heamatococca), Neurospora sp.) (e.g., Neurospora crassa), Penicillium sp., Sporotrichum sp. (e.g., Sporotrichum thermophile), Thielavia sp. (e.g., Thielavia terrestris, Thielavia heterothallica), Trichoderma sp. (e.g., Trichoderma risei)It is either *Verticillium reesei*, etc., or a species of *Verticillium* (e.g., *Verticillium dahliae*).

[0063] In the embodiments, eukaryotic cells include insect cells (e.g., Sf9, Mimic® Sf9, Sf21, High Five® (BT1-TN-5B1-4) or BT1-Ea88 cells), algal cells (e.g., Amphora sp., Bacillariophyceae sp., Dunaliella sp., Chlorella sp., Chlamydomonas sp., Cyanophyta sp. (cyanobacteria), Nannochloropsis sp., Spirulina sp., or Ochromonas sp.) These are cells from plants (e.g., from monocotyledonous plants (e.g., maize, rice, wheat, or foxtail grass (Setaria sp.)) or from dicotyledonous plants (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, false moss (Physcomitrella patens), or Arabidopsis sp.)).

[0064] In this embodiment, the cells are bacteria or prokaryotic cells.

[0065] In the embodiments, prokaryotic cells are Gram-positive cells such as bacillus sp., streptomyces sp., streptococcus sp., staphylococcus sp., or lactobacillus sp. Possible bacillus species include, for example, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus natto, or Bacillus megaterium. In the embodiments, the cells are Bacillus subtilis such as Bacillus 3NA and Bacillus 168. Examples of bacillus species are Bacillus Genetic Stock Center, Biological Sciences 556, 484 West 12. th It can be obtained from Avenue, Columbus, OH 43210-1214.

[0066] In this embodiment, the prokaryotic cells are Gram-negative cells such as Salmonella sp. or Escherichia coli, for example TG1, TG2, W3110, DH1, DHB4, DH5a, HMS174, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue, and Origami, as well as cells derived from E. coli B strains, for example BL-21 or BL21(DE3) or BL21(DE3)pLysS, all of which are commercially available.

[0067] Suitable host cells are commercially available from microbial strain preservation institutions such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or ATCC (American Type Culture Collection).

[0068] In the embodiment, the cell is any one of the cells described herein that comprises a heterogeneous nucleic acid, for example, a recombinant polypeptide, such as a target nucleic acid encoding a recombinant polypeptide selected from Tables 5-8.

[0069] The cells described herein may be cultured according to any method known in the art. In some embodiments, the culture medium lacks amino acids (e.g., one or more of the amino acids listed in Table 1). In embodiments, the culture medium lacks amino acids that can be rescued for biosynthesis if the cells take up the target nucleic acid. In embodiments, cell culture is carried out as batch culture, fed-batch batch culture, simplified fed-batch batch overgrowth (aFOG), draw-and-fill culture, or serial culture. In one embodiment, the cell culture is suspension culture. In one embodiment, the cells or cell culture are placed in vivo for the expression of recombinant polypeptides, for example, in a model organism or human subject.

[0070] In one embodiment, the culture medium does not contain serum. Serum-free, protein-free, and chemically-defined animal component-free (CDACF) media are commercially available, for example, from Lonza Biologics.

[0071] In some embodiments, lipid additives (including, for example, cholesterol, oleic acid, linoleic acid, or combinations thereof) may be added to the culture medium.

[0072] Suitable culture media and culture methods for mammalian cell lines are well known in the art, as described, for example, in U.S. Patent No. 5,633,162. Examples of standard cell culture media for research flasks or low-density cell cultures, and adapted to the requirements of specific cell types, include, for example, Roswell Park Memorial Institute (RPMI) 1640 medium (Morre, G., The Journal of the American Medical Association, 199, p. 519 f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene, 78, p. 173ff. 1963), Dulbecco's modified Eagle's medium (DMEM), Eagle's minimal essential medium (MEM), Ham F12 medium (Ham, R. et al., Proc. Natl. Acad. Sc. 53, p. 288 ff. 1965), or Iscoves modified DMEM without albumin, transferrin, and lecithin (Iscoves et al., J. Exp. med. 1, p. 923 (ff., 1978). For example, Ham F10 or F12 medium was specially designed for CHO cell culture. Other media specially adapted for CHO cell culture are described in EP-481791. Such culture media may be supplemented with fetal bovine serum (FBS (fetal bovine serum), also called fetal calf serum FCS (fetal calf serum)), which is known to provide a natural source of excess hormones and growth factors. Cell culture of mammalian cells is a routine operation that is well described in scientific textbooks and manuals today, and is covered in detail, for example, R. Ian Fresney, Culture of Animal Cells, a manual, 4th edition, Wiley-Liss / NY, 2000. Any of the cell culture media described herein may be formulated to lack certain amino acids (e.g., amino acids listed in Table 1), such as amino acids whose biosynthesis can be rescued if the cell takes up the target nucleic acid.

[0073] Other suitable culture methods are known to those skilled in the art and may depend on the recombinant polypeptide product and the host cells used. Determining or optimizing suitable conditions for the expression and production of recombinant polypeptides or therapeutic polypeptides expressed by cells is within the scope of the art.

[0074] Methods for genetically modifying or manipulating cells to express a desired polypeptide or protein are well known in the art and include, for example, transfection, transduction (e.g., viral transduction), or electroporation of cells with nucleic acids, such as vectors. Examples of physical methods for introducing nucleic acids, such as heterologous nucleic acids or vectors as described herein, into host cells include, but are not limited to, calcium phosphate precipitation, lipofection, particulate guns, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vols. 1-4, Cold Spring Harbor Press, NY). Examples of chemical means for introducing nucleic acids, such as heterologous nucleic acids or vectors as described herein, into host cells include, but are not limited to, colloidal dispersions such as polymer complexes; nanocapsules; microspheres; beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery media in vitro and in vivo include liposomes (e.g., artificial membrane vesicles). Other state-of-the-art methods of nucleic acid targeting delivery, such as delivery of polynucleotides by targeting nanoparticles or other suitable submicron-sized delivery systems, are available.

[0075] nucleic acid Products described herein, such as nucleic acids encoding recombinant polypeptides, such as the target nucleic acid, are also provided herein. Nucleic acid sequences encoding a desired recombinant polypeptide can be obtained using standard techniques, for example, by screening a library of cells expressing the desired nucleic acid sequence, for example, by deriving the nucleic acid sequence from a vector known to contain the desired nucleic acid sequence, or by directly isolating it from cells and tissues containing the desired nucleic acid sequence, or by recombinant methods known in the art. Alternatively, nucleic acids encoding recombinant polypeptides may be produced by synthesis rather than cloning. Recombinant DNA techniques and technologies are highly advanced and well-established in the art. Therefore, those skilled in the art, knowing the amino acid sequences of the recombinant polypeptides described herein, can readily recall or generate nucleic acid sequences capable of encoding recombinant polypeptides.

[0076] Recombinant polypeptide expression is typically achieved by operably ligating a recombinant polypeptide or a nucleic acid encoding a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes or prokaryotes. Typical cloning vectors contain transcriptional and translational terminators, start sequences, and other regulatory elements such as promoters, which are useful for regulating the expression of a desired nucleic acid sequence.

[0077] In embodiments, the product, for example, a heterogeneous therapeutic polypeptide, comprises an antibody or antibody fragment containing multiple polypeptide chains, such as heavy and light chains. The nucleic acid sequences encoding the multiple polypeptide chains may be arranged together (e.g., each polypeptide chain encoding the sequence is located on the same nucleic acid) or separately (e.g., each polypeptide chain encoding the sequence is located on a different nucleic acid). The sequences encoding a heterogeneous therapeutic polypeptide containing multiple polypeptide chains may be operably linked to a single control element, for example, a first control element, or to separate control elements (e.g., each polypeptide chain encoding the sequence is operably linked to its own first control element). In one embodiment in which the sequences encoding a heterogeneous therapeutic polypeptide containing multiple polypeptide chains are operably linked to separate control elements, one or more of the control elements (e.g., 1, 2, 3, 4, 5, 6, or all of them) may have a first level of activity under first conditions and a second level of activity under second conditions, and one or more of the control elements (e.g., 1, 2, 3, 4, 5, 6, or more) may be constitutive.

[0078] Nucleic acid sequences encoding recombinant polypeptides can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including plasmids, phagemids, phage derivatives, animal viruses, and cosmids, without limitation. Vectors for specific purposes include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors. In embodiments, expression vectors may be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vols. 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain a functional origin of replication in at least one organism, a regulatory element including a promoter element and optionally an enhancer element, a convenient restriction endonuclease site, and one or more selectable markers (e.g., International Publication Nos. 01 / 96584, 01 / 29058, and U.S. Patent No. 6,326,193). Viral vectors are suitable tools for achieving long-term gene transfer because they enable the long-term stable integration and growth of the transgene in daughter cells.

[0079] The vector may also include, for example, signal sequences that promote secretion, polyadenylation signals and transcription terminators (e.g., from the bovine growth hormone (BGH) gene), elements that enable episomal replication and replication in prokaryotes (e.g., the SV40 origin and ColE1 or others known in the art), and / or elements that enable selection, such as selection markers or reporter genes.

[0080] The intended vector may include an insertion site suitable for inserting a sequence encoding a polypeptide, such as an exogenous therapeutic polypeptide or a repressor polypeptide.

[0081] The insertion site may include a restriction endonuclease site.

[0082] The insertion site may include a recombinant target site, which is adjacent to a sequence encoding a polypeptide, such as an exogenous therapeutic polypeptide or a repressor polypeptide. In one embodiment, the recombinant target site is a lox site. When the recombinant target site is a lox site, the host cell requires the presence and expression of Cre recombinase to achieve crossover or recombination events.

[0083] In embodiments, a vector comprising a nucleic acid sequence encoding a product described herein, such as a polypeptide, such as a recombinant polypeptide, further comprises a nucleic acid sequence encoding a selection marker. In embodiments, the selection marker includes glutamine synthetase (GS); dihydrofolate reductase (DHFR), such as an enzyme conferring resistance to methotrexate (MTX); proline; or an antibiotic marker, such as an enzyme conferring resistance to antibiotics such as hygromycin, neomycin (G418), zeosin, puromycin, or blastocydin. In embodiments, the selection marker includes or is compatible with Selexis selection systems (e.g., SUREtechnology Platform® and Selexis Genetic Elements®, commercially available from Selexis SA) or Catalant selection systems.

[0084] In some embodiments, a vector containing a nucleic acid sequence encoding a recombinant product as described herein includes a selection marker useful for identifying one or more cells containing the nucleic acid encoding the recombinant product as described herein. In another embodiment, the selection marker is useful for identifying one or more cells containing the integration of the nucleic acid sequence encoding a recombinant product into the genome, as described herein. Identifying one or more cells containing the integration of the nucleic acid sequence encoding a recombinant protein may be useful for selecting and manipulating cells or cell lines that stably express the product.

[0085] product Compositions and methods for identifying, selecting, or culturing cells or cell lines capable of producing high-yield products, such as polypeptides, such as therapeutic polypeptides, are provided herein. Products encompassed by this disclosure include, but are not limited to, molecules, nucleic acids (e.g., non-coding nucleic acids, e.g., non-coding RNA molecules, e.g., antisense RNA, siRNA, tRNA, ribosomal RNA, microRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, or long non-coding RNA, e.g., Xist or Hotair), polypeptides (e.g., recombinant polypeptides and / or therapeutic polypeptides), or hybrids thereof, which may be produced by expression in cells. In some embodiments, cells are manipulated or modified to produce products. Such modifications include the introduction of molecules that control or result in product production. For example, cells are modified by introducing heterologous nucleic acids encoding polypeptides, e.g., recombinant polypeptides, and the cells are cultured under conditions suitable for the production, e.g., expression and secretion, of polypeptides, e.g., recombinant polypeptides. In another example, cells are modified by introducing heterologous nucleic acids that control, for example, increase, the expression of polypeptides endogenously expressed by the cell, so that the cells produce higher levels or amounts of polypeptides than, for example, those endogenously produced in unmodified cells. In embodiments, cells or cell lines identified, selected, or produced by the methods described herein produce products useful in the treatment of health conditions, disorders, or diseases, such as recombinant polypeptides. Examples of health conditions, disorders, or diseases include, but are not limited to, metabolic disorders or disorders (e.g., metabolic enzyme deficiencies), endocrine disorders (e.g., hormone deficiencies), hemostasis, thrombosis, hematopoietic disorders, pulmonary disorders, gastrointestinal disorders, immunomodulation (e.g., immune deficiencies), infertility, transplantation, cancer, and infectious diseases.

[0086] Polypeptide In some embodiments, the product is a polypeptide, such as a recombinant polypeptide. The polypeptide product may be used in any of the compositions (e.g., cells) and methods described herein in some embodiments. In some embodiments, the polypeptide is produced by cells grown in a medium containing an inhibitor, such as an enzyme molecule, as described herein. In some embodiments, the polypeptide is produced by cells containing LMM, as described herein. In embodiments, LMM is expressed under the control of a promoter, as described herein. In some embodiments, the polypeptide is produced by cells grown in a medium containing LMM, as described herein.

[0087] In the embodiments, the polypeptide is a heterologous polypeptide, such as a heterologous protein, such as a protein not naturally expressed by cells. The polypeptide may be a therapeutic or diagnostic protein, for example, useful for drug screening. The therapeutic or diagnostic protein may be an antibody molecule, such as an antibody or antibody fragment, a fusion protein, a hormone, a cytokine, a growth factor, an enzyme, a glycoprotein, a lipoprotein, a reporter protein, a therapeutic peptide, an aptamer or structural and / or functional fragment, or a hybrid of any of these. In the embodiments, the product, such as a heterologous therapeutic polypeptide, includes an antibody or antibody fragment comprising multiple polypeptide chains, such as heavy and light chains.

[0088] In some embodiments, the product, for example, a recombinant polypeptide, is an antibody molecule. Products incorporated herein include diagnostic antibody molecules useful for imaging techniques, such as monoclonal antibodies or antibody fragments thereof, and therapeutic antibody molecules suitable for administration to a subject, for example, useful for treating a disease or disorder. The antibody molecule is a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. In one embodiment, the antibody molecule is a full-length antibody or antibody fragment. The antibody and polymorphic protein may be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulin, and may be derived from a natural source or a recombinant source. The antibody may be a tetramer of an immunoglobulin molecule. In one embodiment, the antibody is a monoclonal antibody. The antibody may be human or a humanized antibody. In one embodiment, the antibody is an IgA, IgG, IgD, or IgE antibody. In one embodiment, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0089] An "antibody fragment" refers to at least a portion of an intact antibody, or a recombinant variant thereof, and refers to the antigen-binding domain of the intact antibody, such as the antigen-determining variable region, which is sufficient to give the antibody fragment recognition and specific binding to a target such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camel VHH domains, and bivalent fragments containing two Fab fragments linked by disulfide crosslinking at the hinge region, as well as isolated CDRs or other epitope-binding fragments of antibodies. Furthermore, antigen-binding fragments can be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intracellular antibodies, bispecific antibodies, tripspecific antibodies, quadruplespecific antibodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Additionally, antigen-binding fragments can be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Patent No. 6703199, which describes fibronectin polypeptide minibodies).

[0090] In embodiments, polypeptides (e.g., polypeptides produced by cells and / or polypeptides produced according to the methods described herein) include, for example, Botox, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxin), alglucosidase alfa, daptomycin, YH-16, chorionic gonadotropin alfa, filgrastim, cetrorelix, interleukin-2, aldesleukin, teceleulinin, denileukin difutitox, interferon alfa-n3 (injection), interferon alfa-n1, DL-8234, interferon, Suntory (gamma-1a), interferon gamma, thymosin alfa-1, tasonelmin, and Dizzy DigiFab, ViperaTAb, EchiTAb, CroFab, Nesiritide, Abatacept, Alefacept, Rebif, Eptothermin Alpha, Teriparatide, Calcitonin, Etanercept, Hemoglobin Glutamer 250 (Bovine), Drotrecogin Alpha, Collagenase, Carperitide, Recombinant Human Epidermal Growth Factor, DWP401, Darbe Poetin alpha, epoetin omega, epoetin beta, epoetin alpha, decilidine, repiridine, vivalilidine, nonacog alpha, mononine, eptacog alpha (activated), recombinant factor VIII + VWF, Recombinate, recombinant factor VIII, factor VIII (recombinant), Alphanmate, octocog alpha alpha), factor VIII, palifermin, indikinase, tenecteplase, alteplase, pamiteplase, reteplase, nateplase, monteplase, follitropin alpha, rFSH, hpFSH, micafungin, pegfilgrastim, lenograstim, naltograstim, selmorelin, glucagon, exenatide, pramlintide, imiglucerase, galsulfase, leucotropin,Morgramostirn, Triptorelin acetate, Histrelin (Hydron), Deslorerin, Histrelin, Nafarelin, Leuprolide (ATRIGEL), Leuprolide (DUROS), Goserelin, Eutropin, Somatropin, Mecasermin, Enlfavirtide, Org-33408, Insulin glargine, Insulin glulisine, Ins Phosphate (inhalant), insulin lispro, insulin deternir, insulin (RapidMist), mecasermin lymphabate, anakinra, celemolukin, 99mTc-apsitide, myelopide, betaseron, glatiramer acetate, Gepon, salglamostim, oprelbequin, human leukocyte-derived alpha interferon, bilive, insulin (recombinant), recombinant human Insulin, insulin aspart, mecasenin, Roferon-A, interferon-alpha-2, alfaferone, interferon alfacon-1, interferon alfa, Avonex recombinant human luteinizing hormone, dorunase alfa, trafermin, diconotide, taltirelin, dibotermin alfa, atosiban, becaprelmin, eptifibatide, zemyla (Z Emaira), CTC-111, Shanvac-B, Octreotide, Lanreotide, Ancestim, Agalsidase Beta, Agalsidase Alpha, Laronidase, Prezatide Copper Acetate, Rasburicase, Ranibizumab, Actimmune, PEG-Intron, Tricomin, Recombinant Human Parathyroid Hormone (PTH) 1-84, Epoetin Delta, Transgenic Antithrombin III, Granditropin, Vitrase, Recombinant Insulin, Interferon-Alpha, GEM-21S, Vapreotide, Idursulfase,Omnapatrilat, recombinant serum albumin, certolizumab pegol, glucarpidase, human recombinant C1 esterase inhibitor, lanoteplase, recombinant human growth hormone, enfuvirtide, VGV-1, interferon (alpha), lucinactant, aviptadil, icatibant, ecalantide, omiganan, aurograb, pexiganan acetate, ADI-PEG-20, LDI-200, degarelix, cintredelinbesudotox ox), Favld, MDX-1379, ISAtx-247, liraglutide, teriparatide, tifacogin, AA4500, T4N5 liposome lotion, catsumakisomab, DWP413, ART-123, Chrysalin, desmoteplase, amediplase, corifolitropin alfa, TH-9507, teduglutide, Diamyd, DWP-412, growth hormone, recombinant G-CSF, insulin, insulin (Technosphere (Techno sphere), insulin (AERx), RGN-303, DiaPep 277, interferon beta, interferon alpha-n3, belatacept, transdermal insulin patch, AMG-531, MBP-8298, Xerecept, opebacan, AIDSVAX, GV-1001, LymphoScan, lampirase, lipoxysan, rusplutide, MP52, ciproisel-T, CTP-37, Insegia, Vitespen, Human Thrombin, Thrombin, TransMID, Alfimeprase, Puricase, Terlipressin, EUR-1008M, Recombinant FGF-I, BDM-E, Rotigaptide, ETC-216, P-113, MBI-594AN, Duramycin, SCV-07, OPI-45, Endostatin, Angiostatin, ABT-510, Bowman-Birk inhibitor, XMP-629,99mTc-Hynic-Annexin V, Kahalalid F, CTCE-9908, Teverelix, Ozarelix, Rornidepsin, BAY-504798, Interleukin 4, PRX-321, Pepscan, Ivocatekin, Rh lactoferrin, TRU-015, IL-21, ATN-1 61, Silenditide, Albuferon, Biphasix, IRX-2, Omega Interferon, PCK-3145, CAP-232, Pasireotide, huN901-DMI, SB-249553, Oncovax-CL, Oncovax-P, BLP-25, CerVax-16, MAR T)-1, gp100, tyrosinase, nemificide, rAAT, CGRP, pegsnercept, thymosin beta-4, plitidepsin, GTP-200, lamoplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (eligen), examorelin, capromorelin, Cardeva, berafermin, 131I-TM-601, KK-220, T-10, uralitide, depelestat, hematide, chrysalin, rNAPc2, recombinant factor VIII (PEGylated liposome), bFGF, PEGylated recombinant staphylokinase variant, V-10153, SonoLysis Prolyze Prolyse), NeuroVax, CZEN-002, rGLP-1, BIM-51077, LY-548806, Exenatide (controlled release, Medisorb), AVE-0010, GA-GCB, Avorelin, ACM-9604, Linaclotide acetate, CETi-1, Hemospan, VAL, Rapid-acting insulin (for injection, Viadel), Insulin (Erigen), Recombinant methionyl human leptin, Pitraquinra, Multikine, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141,Cpn10, talactoferrin, rEV-131, rEV-131, recombinant human insulin, RPI-78M, oprelbequin, CYT-99007 CTLA4-Ig, DTY-001, valategrast, interferon alpha-n3, IRX-3, RDP-58, tauferon, bile salt-stimulating lipase, merispase, alkaline phosphatase, EP-2104R, melanotan-II, bremelanotide, ATL-104, recombinant human microplasmin, AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, dynorphin A, SI-6603, LAB GHRH, AER-002, BGC-728, ALTU-135, Recombinant Neuraminidase, Vacc-5q, Vacc-4x, Tat Toxoid, YSPSL, CHS-13340, PTH(1~34)(Novasome), Ostabolin-C, PTH Analogue, MBRI-93.02, MTB72F, MVA-Ag85A, FARA04, BA-210, Recombinant Plague FIV, AG-702, OxSODrol, rBetV1, Der-p1 / Der-p2 / Der-p7, PR1 Peptide Antigen, Mutant Ras Vaccine, HPV-16 E7 lipopeptide vaccine, labyrinthin, WT1-peptide, IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, icrocaptide, telbermin, lupintrivir, reticulose, rGRF, HA, alpha-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensin, D-4F, ETC-642, APP-018, rhMBL, SCV-07, DRF-7295, ABT-828, ErbB2-specific immunotoxin, DT3SSIL-3, TST-10088, PRO-1762, Combotox,Cholecystokinin-B / gastrin receptor binding peptide, 111In-hEGF, AE-37, trastuzumab-DM1, antagonist G, IL-12, PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647, L-19 based ra, Re-188-P-2045, AMG-386, DC / 1540 / , KLH, VX-001, AVE-9633, AC-9301, NY-ESO-1 (peptide), NA17.A2 peptide, CBP-501, recombinant human lactoferrin, FX-06, AP-214, WAP-8294A, ACP-HIP, SUN-11031, peptide YY[3~36], FGLL, Atasicept, BR3-Fc, BN-003, BA-058, human parathyroid hormone 1~34, F-18-CCR1, AT-1100, JPD-003, PTH(7~34)(N ovasome), duramycin, CAB-2, CTCE-0214, GlycoPEGylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, Factor XIII, aminocandin, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, teverelix, EP-51216, hGH, OGP-I, shifvirtide, TV4710, ALG-889, Org-41259, rh CC10, F-991, thymopentin, r(m)CRP, hepatic selective insulin, subalin, L19-IL-2 fusion protein, elaphin, NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist, AL-108, AL-208, nerve growth factor antagonist, SLV-317, CGX-1007, INNO-105, teriparatide (Erigen), GEM-OS1, AC-162352, PRX-302, These include LFn-p24 fusion, EP-1043, gpE1, gpE2, MF-59, hPTH(1~34), 768974, SYN-101, PGN-0052, abiscumin, BIM-23190, polyepitope tyrosinase peptide, enkastim, APC-8024, GI-5005, ACC-001, TTS-CD3, vascular targeting TNF, desmopressin, onercept, and TP-9201.

[0091] In some embodiments, the polypeptide (e.g., a polypeptide produced by cells and / or a polypeptide produced according to the methods described herein) is any other suitable polypeptide, including adalimumab (Humira), infliximab (Remicade), rituximab (Rituxan / Mab Thera), etanercept (Enbrel), bevacizumab (Avastin), trastuzumab (Herceptin), pegfilgrastim (Neulasta), or any other suitable polypeptide, including biosimilars and biomodified products.

[0092] Other suitable polypeptides (e.g., polypeptides produced by cells and / or polypeptides produced according to the methods described herein) include, but are not limited to, those listed in Table 5 below and in Table 1 of U.S. Patent Application Publication No. 2016 / 0097074.

[0093] [Table 5] TIFF2026048662000018.tif211149 TIFF2026048662000019.tif210149 TIFF2026048662000020.tif211149 TIFF2026048662000021.tif143149

[0094] In embodiments, polypeptides (e.g., polypeptides produced by cells and / or polypeptides produced according to the methods described herein) are hormones, coagulation / blood clotting factors, cytokines / growth factors, antibody molecules, fusion proteins, protein vaccines, or peptides, as shown, for example, in Table 6.

[0095] [Table 6] TIFF2026048662000023.tif226149 TIFF2026048662000024.tif206149 TIFF2026048662000025.tif103149

[0096] In the embodiments, the polypeptide (e.g., a polypeptide produced by a cell and / or a polypeptide produced according to the methods described herein) is a multispecific protein, such as a bispecific antibody. In the embodiments, the multispecific proteins are as shown in Table 7.

[0097] [Table 7] TIFF2026048662000027.tif157149 TIFF2026048662000028.tif163149 TIFF2026048662000029.tif165149 TIFF2026048662000030.tif153149 TIFF2026048662000031.tif147149 TIFF2026048662000032.tif152149 TIFF2026048662000033.tif158149 TIFF2026048662000034.tif163149 TIFF2026048662000035.tif104149

[0098] In the embodiments, the polypeptide (e.g., a polypeptide produced by a cell and / or a polypeptide produced according to the methods described herein) is one of the polypeptides listed in Table 8.

[0099]

Table 8

[0100] In some embodiments, the polypeptide (e.g., a polypeptide produced by cells and / or a polypeptide produced according to the methods described herein) is an antigen expressed by cancer cells. In some embodiments, the recombinant polypeptide or therapeutic polypeptide is a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the recombinant polypeptide or therapeutic polypeptide is HER2, CD20, 9-O-acetyl-GD3, βhCG, A33 antigen, CA19-9 marker, CA-125 marker, calreticulin, carbonic anhydrase IX (MN / CA IX), CCR5, CCR8, CD19, CD22, CD25, CD27, CD30, CD33, CD38, CD44v6, CD63, CD70, CC123, CD138, carcinoma embryonic antigen (CEA; CD66e), desmoglein 4, E-cadherin neoepitope, endothialin, ephrin A2 (EphA2), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM). (molecule), ErbB2, fetal acetylcholine receptor, fibroblast activation antigen (FAP), fucosyl GM1, GD2, GD3, GM2, ganglioside GD3, globo H, glycoprotein 100, HER2 / neu, HER3, HER4, insulin-like growth factor receptor 1, Lewis-Y, LG, Ly-6, melanoma-specific chondroitin-sulfate proteoglycan (MCSCP), mesoserine, MUC1, MUC2, MUC3, MUC4, MUC5 AC MUC5 B The following are selected: MUC7, MUC16, Müllerian inhibitory substance (MIS) receptor type II, plasma cell antigens, polySA, PSCA, PSMA, sonic hedgehog (SHH), SAS, STEAP, sTn antigen, TNF-alpha precursor, and combinations thereof.

[0101] In some embodiments, polypeptides (e.g., polypeptides produced by cells and / or polypeptides produced according to the methods described herein) are activating receptors, including 2B4 (CD244), α4β1 integrin, β2 integrin, CD2, CD16, CD27, CD38, CD96, CD100, CD160, CD137, CEACAM1 (CD66), CRTAM, CS1 (CD319), DNAM-1 (CD226), GITR (TNFRSF18) ), selected from activated forms of KIR, NKG2C, NKG2D, NKG2E, one or more native cytotoxic receptors, NTB-A, PEN-5, and combinations thereof, optionally comprising β2 integrins CD11a-CD18, CD11b-CD18, or CD11c-CD18, optionally comprising activated forms of KIR KIR KIR2DS1, KIR2DS4, or KIR-S, and optionally comprising native cytotoxic receptors NKp30, NKp44, NKp46, or NKp80.

[0102] In some embodiments, the polypeptide (e.g., a polypeptide produced by a cell and / or a polypeptide produced according to the methods described herein) is an inhibitory receptor selected from KIR, ILT2 / LIR-1 / CD85j, inhibitory forms of KIR, KLRG1, LAIR-1, NKG2A, NKR-PIA, Siglec-3, Siglec-7, Siglec-9 and combinations thereof, and optionally the inhibitory forms of KIR include KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2 or KIR-L.

[0103] In some embodiments, polypeptides (e.g., polypeptides produced by cells and / or polypeptides produced according to the methods described herein) are activating receptors, and include CD3, CD2 (LFA2, OX34), CD5, CD27 (TNFRSF7), CD28, CD30 (TNFRSF8), CD40L, CD84 (SLAMF5), CD137 (4-1BB), CD226, CD229 (Ly9, SLAMF3), CD244 (2B4, SLAMF 4) Selected from CD319 (CRACC, BLAME), CD352 (Ly108, NTBA, SLAMF6), CRTAM (CD355), DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS, LIGHT, LTβR (TNFRSF3), OX40 (CD134), NKG2D, SLAM (CD150, SLAMF1), TCRα, TCRβ, TCRδγ, TIM1 (HAVCR, KIM1), and combinations thereof.

[0104] In some embodiments, the polypeptide (e.g., a polypeptide produced by cells and / or a polypeptide produced according to the methods described herein) is an inhibitory receptor and is selected from PD-1 (CD279), 2B4 (CD244, SLAMF4), B71 (CD80), B7H1 (CD274, PD-L1), BTLA (CD272), CD160 (BY55, NK28), CD352 (Ly108, NTBA, SLAMF6), CD358 (DR6), CTLA-4 (CD152), LAG3, LAIR1, PD-1H (VISTA), TIGIT (VSIG9, VSTM3), TIM2 (TIMD2), TIM3 (HAVCR2, KIM3), and combinations thereof.

[0105] Other exemplary polypeptides (e.g., polypeptides produced by cells and / or polypeptides produced according to the methods described herein) include, but are not limited to, any proteins listed in Tables 1-10 of Leader et al., "Protein therapeutics: a summary and pharmacological classification," Nature Reviews Drug Discovery, 2008, 7:21-39 (incorporated herein by reference); or any conjugates, variants, analogs, or functional fragments of recombinant polypeptides described herein.

[0106] Other recombinant protein products (e.g., products produced by cells and / or products produced according to the methods described herein) include, but are not limited to, non-antibody scaffolds or scaffold protein substitutes such as DARPin, affibody, and adnectin. Such non-antibody scaffolds or scaffold protein substitutes may be engineered to recognize or bind to one, two, or more, for example, one, two, three, four, or five or more different targets or antigens.

[0107] Applicable The methods for identifying, selecting, and / or culturing cells disclosed herein may be used to generate cells useful for producing various products, to evaluate various cell lines, or to evaluate the production of various cell lines for use in bioreactors or processing vessels or tanks, or more generally, for use by any source. The compositions and methods described herein are suitable for culturing any desired cell line, including, for example, prokaryotic cell lines and / or eukaryotic cell lines. Furthermore, in embodiments, the compositions and methods described herein are suitable for culturing suspension cells or anchorage-dependent (adherent) cells and are suitable for production operations configured for the production of pharmaceutical and biopharmaceutical products such as polypeptide products, nucleic acid products (e.g., DNA or RNA), or cells and / or viruses used in cell therapy and / or viral therapy.

[0108] In embodiments, cells express or produce products such as recombinant therapeutic products or diagnostic products. Examples of products produced by cells, as will be described in detail below, include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (e.g., polypeptide molecules that specifically bind to antigens but are not structurally related to antibodies, such as DARPin, aphidophors, adnectin, or IgNAR), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapeutics (e.g., anti-cancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cell therapeutics (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), vaccines or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA, etc.) or DNA (e.g., plasmid DNA, etc.), antibiotics, or amino acids. In embodiments, the compositions and methods described herein may be used to produce biosimilars.

[0109] As mentioned herein, in embodiments, the compositions and methods described herein enable the production of eukaryotic cells, such as mammalian cells, or lower eukaryotic cells such as yeast cells or filamentous fungal cells, or prokaryotic cells such as Gram-positive or Gram-negative cells, and / or the synthesis of eukaryotic or prokaryotic cell products, such as proteins, peptides, antibiotics, amino acids, nucleic acids (DNA or RNA, etc.), by eukaryotic cells on a large scale. Unless otherwise specified herein, the compositions and methods described herein may include any desired volume or production capacity, non-limitingly including bench-scale, pilot-scale, and full-scale production volumes.

[0110] Furthermore, unless otherwise specified herein, the compositions and methods described herein may be used with any suitable reactor(s), including but not limited to agitated tanks, bubble pumps, fibers, microfibers, hollow fibers, ceramic matrices, fluidized beds, fixed beds and / or jet bed bioreactors. As used herein, “reactor” may include a fermenter or fermentation unit or any other reaction vessel, and the term “reactor” is used interchangeably with “fermenter.” For example, in some embodiments, a bioreactor unit may perform one or more or all of the following: supplying nutrients and / or carbon sources, injecting suitable gases (e.g., oxygen), fermentation or inlet / outlet fluidization of cell culture media, separation of gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, stirring (e.g., agitation), and / or cleaning / sterilization. Examples of reactor units, such as fermentation units, may contain multiple reactors within the unit, for example, a unit having 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors in each unit, and / or a facility may contain multiple units, each having one or multiple reactors within the facility. In various embodiments, the bioreactor may be suitable for batch, semi-flow batch, flow batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor may have a volume of about 100 mL to about 50,000 L.Non-restrictive examples include 100mL, 250mL, 500mL, 750mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, and 550 liters. Volumes include 10 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. In addition, preferred reactors may be multi-use, single-use, disposable, or non-disposable, and may be formed from any preferred material including stainless steel (e.g., 316L or any other preferred stainless steel) and metal alloys such as Inconel, plastics, and / or glass. In some embodiments, preferred reactors may be circular, for example, cylindrical. In some embodiments, a suitable reactor may be rectangular, for example. Rectangular reactors may, in some cases, offer advantages over circular reactors, such as ease of use (e.g., loading and setup by technicians), greater mixing and uniformity of the reactor contents, and a smaller floor area occupied.

[0111] In embodiments, and unless otherwise indicated herein, the compositions and methods described herein may be used by any suitable unit operations and / or equipment not otherwise mentioned, such as operations and / or equipment for the separation, purification and isolation of such products. Any suitable facilities and environments may be used, such as traditional field-assembled facilities, modular, mobile and temporary facilities or any other suitable structures, facilities and / or layouts. For example, in some embodiments, a modular cleanroom may be used. In addition, and unless otherwise indicated herein, the compositions and methods described herein may be housed and / or implemented in a single location or facility, or in separate or multiple locations and / or facilities.

[0112] As non-limiting examples, and not limiting, U.S. Patent Publications 2013 / 0280797, 2012 / 0077429, 2011 / 0280797, 2009 / 0305626, and U.S. Patents 8,298,054, 7629,167, and 5656491 describe exemplary equipment, apparatus and / or systems that may be suitable for use in the compositions and methods described herein, and such patents and patent applications are incorporated herein by reference in their entirety.

[0113] The compositions and methods described herein can utilize a broad spectrum of cells. In embodiments, the cells are eukaryotic cells, such as mammalian cells. Mammalian cells may be, for example, human, rodent, or bovine cell lines or cell lines. Examples of such cells, cell lines, or cell lines include, for example, mouse myeloma (NS0) cell line, Chinese hamster ovary (CHO) cell line, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, such as COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EB1, EB2, EB3, oncolytic cell lines, or hybridoma cell lines. Preferably, the mammalian cells are CHO cell lines. In one embodiment, the cells are CHO cells. In one embodiment, the cells are CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN, or CHO-derived cells. CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1 SV GS knockout cells. CHO FUT8 knockout cells are, for example, Potelligent® CHOK1 SV (Lonza Biologics, Inc.). Eukaryotic cells may be, for example, avian cells, cell lines, or cell lines such as EBx® cells, EB14, EB24, EB26, EB66, or EBv13.

[0114] In one embodiment, eukaryotic cells are stem cells. Stem cells may be pluripotent stem cells, including, for example, embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs).

[0115] In one embodiment, the cells are differentiated forms of any of the cells described herein. In one embodiment, the cells are cells derived from any primary cells in a culture.

[0116] In the embodiments, the cells are hepatocytes such as human hepatocytes, animal hepatocytes, or non-parenchymal cells. For example, the cells may be seedable metabolic human hepatocytes, seedable induction human hepatocytes, seedable qualist transporter certified® human hepatocytes, human hepatocytes for suspension (including pooled hepatocytes from 10 and 20 donors), human hepatic Kupffer cells, human hepatic stellate cells, canine hepatocytes (including single and pooled beagle hepatocytes), mouse hepatocytes (including CD-1 and C57Bl / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including cynomolgus or rhesus monkey hepatocytes), feline hepatocytes (including domestic cat shorthair hepatocytes), and rabbit hepatocytes (including New Zealand white hepatocytes). The liver cells in question are commercially available from Triangle Research Labs, LLC, 6 Davis Drive, Research Triangle Park, North Carolina, USA 27709.

[0117] In one embodiment, the eukaryotic cells are lower eukaryotic cells such as yeast cells (e.g., Pichia genera (e.g., Pichia pastris, Pichia metanorica, Pichia cruibergii and Pichia angusta), Chomagataera genera (e.g., Chomagataera pastris, Chomagataera pseudopastoris or Chomagataera fafi), Saccharomyces genera (e.g., budding yeast, budding yeast, Saccharomyces cruibergii, Saccharomyces ubaram), Cruiveromyces genera (e.g., Cruiveromyces lactis, Cruiveromyces marxianus), Candida genera (e.g., Torula yeast, Candida cacaoi, Candida boidini), Geotrichum genera (e.g., Geotrichum fermentans), Hansenula polymorpha, Yarrowia liporitica or fission yeast). Pichia pastris varieties are preferred. Examples of Pichia pastris strains include X33, GS115, KM71, KM71H, and CBS7435.

[0118] In one embodiment, eukaryotic cells are fungal cells (e.g., Aspergillus species (Aspergillus niger, Aspergillus fumigatus, Aspergillus japonica, Pseudosomal Aspergillus, etc.), Acremonium species (Acremonium thermophyllum, etc.), Cetoma species (Cetoma thermophyllum, etc.), Chrysosporium species (Chrysosporium thermophyllum, etc.), Cordyceps species (Cordyceps, etc.), Corinascus species, Ctenomyces species, Fusarium species (Fusarium oxysporum, etc.), Glomerella species (Poaceae anthracnose fungi, etc.), and Peony fungus These include genera such as Hypoclea jecolina, Magnaporte (rice blast fungus, etc.), Myceliophthora (Myceliophthora thermophil, etc.), Nectaria haematococca (Nectaria haematococca, etc.), Neurospora crassa (Nepaleseed crassa, etc.), Penicillium, Sporotricum (Sporotricum thermophil, etc.), Tierrabia (Tierrabia terrestris, Tierrabia heterotalica, etc.), Trichoderma (Trichoderma risei, etc.), or Verticillium (Verticillium darie, etc.).

[0119] In one embodiment, the eukaryotic cells are insect cells (e.g., Sf9, Mimic® Sf9, Sf21, High Five® (BT1-TN-5B1-4) or BT1-Ea88 cells), algal cells (e.g., cells of the genera Amphora, diatoms, Dunaliella, Chlorella, Euglena, cyanobacteria, Nannochloropsis, Spirulina, or Ochromonas), or plant cells (e.g., cells from monocots (e.g., maize, rice, wheat, or foxtail) or dicots (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, false bellflower, or Arabidopsis)).

[0120] In embodiments, cultured cells are used to produce proteins, such as antibodies, such as monoclonal antibodies and / or recombinant proteins, for therapeutic use. In embodiments, cultured cells produce peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites. For example, in embodiments, molecules having molecular weights of about 4,000 daltons to over 140,000 daltons may be produced. In embodiments, these molecules may have some complexity and may include post-translational modifications, including glycosylation.

[0121] Numbered Embodiments The present invention can be defined, for example, as defined in any of the following numbered paragraphs. 1. A method for identifying, selecting, or culturing cells containing a target nucleic acid sequence, a)(i) Target nucleic acid sequence and (ii) When expressed, nucleic acid sequences that result in high levels of activity of enzymes in amino acid synthesis pathways, such as the proline synthesis pathway, for example, nucleic acid sequences that encode enzyme molecules containing activity A step of preparing cells containing heterogeneous nucleic acids, such as vectors, such as replicable vectors or integration vectors, b) A step of culturing cells containing nucleic acid sequences in a medium containing amino acids, such as proline, at levels insufficient to support the growth of cells that are the same as cells that do not have high activity, under conditions sufficient to allow the growth of cells containing nucleic acid sequences. A method for identifying, selecting, or culturing cells containing heterogeneous nucleic acid sequences, including the above.

[0122] 2. The method according to Embodiment 1, wherein the vector contains heterogeneous nucleic acids.

[0123] 3. The method according to Embodiment 1 or 2, wherein the heterogeneous nucleic acid includes a replicable vector, such as a self-replicating vector.

[0124] 4. The method according to any one of the embodiments, wherein heterogeneous nucleic acids include an integration vector.

[0125] 5. The method according to any one of the embodiments, wherein the culture medium further comprises an inhibitor of enzyme activity.

[0126] 6. The method according to Embodiment 5, wherein the inhibitor is an amino acid.

[0127] 7. The method according to Embodiment 5, wherein the inhibitor is not an amino acid.

[0128] 8. A method according to any one of the embodiments, comprising the step of identifying cells containing heterogeneous nucleic acid sequences.

[0129] 9. A method according to any one of the embodiments, comprising the step of selecting cells containing heterogeneous nucleic acid sequences.

[0130] 10. The method according to any one of the embodiments, comprising the step of culturing cells containing heterologous nucleic acid sequences.

[0131] 11. The method according to any one of the embodiments, wherein heterogeneous nucleic acids are included in multiple vectors (for example, the nucleic acids that yield high levels of activity of the target nucleic acid and enzyme are included in multiple vectors, for example, in different vectors).

[0132] 12. The method according to any one of the embodiments, wherein heterogeneous nucleic acids are included in multiple integration vectors (for example, the nucleic acids that result in high levels of activity of the target nucleic acid and enzyme are included in multiple integration vectors, for example, in different vectors).

[0133] 13. The method according to any one of the embodiments, wherein heterogeneous nucleic acids are included in multiple self-replicating vectors (for example, the nucleic acids that result in high levels of activity of the target nucleic acid and enzyme are included in multiple self-replicating vectors, for example, in different vectors).

[0134] 14. The method according to any one of the embodiments, wherein different nucleic acids are integrated into the genome of a cell, for example, a chromosomal genome.

[0135] 15. The method according to any one of the embodiments, wherein the amino acid includes a natural amino acid.

[0136] 16. The method according to any one of the embodiments, wherein the amino acid comprises the amino acids listed in Table 1.

[0137] 17. The method according to Embodiment 16, wherein the amino acid is alanine.

[0138] 18. The method according to Embodiment 16, wherein the amino acid is leucine.

[0139] 19. The method according to Embodiment 16, wherein the amino acid is isoleucine.

[0140] 20. The method according to Embodiment 16, wherein the amino acid is methionine.

[0141] 21. The method according to Embodiment 16, wherein the amino acid is valine.

[0142] 22. The method according to Embodiment 16, wherein the amino acid is phenylalanine.

[0143] 23. The method according to Embodiment 16, wherein the amino acid is asparagine.

[0144] 24. The method according to Embodiment 16, wherein the amino acid is cysteine.

[0145] 25. The method according to Embodiment 16, wherein the amino acid is glutamine.

[0146] 26. The method according to Embodiment 16, wherein the amino acid is serine.

[0147] 27. The method according to Embodiment 16, wherein the amino acid is threonine.

[0148] 28. The method according to Embodiment 16, wherein the amino acid is aspartic acid.

[0149] 29. The method according to Embodiment 16, wherein the amino acid is glutamic acid.

[0150] 30. The method according to Embodiment 16, wherein the amino acid is arginine.

[0151] 31. The method according to Embodiment 16, wherein the amino acid is histidine.

[0152] 32. The method according to Embodiment 16, wherein the amino acid is lysine.

[0153] 33. The method according to Embodiment 16, wherein the amino acid is glycine.

[0154] 34. The method according to any one of Embodiments 1 to 16, wherein the amino acid is selected from proline, tyrosine, and tryptophan.

[0155] 35. The method according to Embodiment 34, wherein the amino acid is proline.

[0156] 36. The method according to Embodiment 34, wherein the amino acid is tyrosine.

[0157] 37. The method according to Embodiment 34, wherein the amino acid is tryptophan.

[0158] 38. The method according to any one of Embodiments 5 to 37, wherein the inhibitor binds to an enzyme, for example, the inhibitor binds to an enzyme and inhibits it.

[0159] 39. The method according to any one of embodiments 5 to 38, wherein the inhibitor inhibits the transcription of the enzyme.

[0160] 40. The method according to any one of embodiments 5 to 39, wherein the inhibitor inhibits the translation of the enzyme.

[0161] 41. The method according to any one of Embodiments 5 to 40, wherein the inhibitor comprises nucleic acids, such as RNA, such as antisense or siRNA.

[0162] 42. The method according to any one of Embodiments 5 to 41, wherein the inhibitor comprises an aptamer.

[0163] 43. The method according to any one of Embodiments 5 to 42, wherein the inhibitor comprises a small molecule.

[0164] 44. The method according to any one of Embodiments 5 to 43, wherein the inhibitor is an analog of the enzyme substrate.

[0165] 45. The method according to any one of Embodiments 5 to 44, wherein the inhibitor is an analog of an amino acid, such as an analog of proline, tyrosine, or tryptophan.

[0166] 46. ​​The method according to any one of Embodiments 5 to 45, wherein the inhibitor includes a competitive inhibitor.

[0167] 47. The method according to any one of Embodiments 5 to 46, wherein the inhibitor inhibits the rate-limiting enzyme for amino acid synthesis, for example, the inhibitor inhibits the rate-limiting enzyme for amino acid synthesis in the culture medium.

[0168] 48. The method according to any one of the embodiments, wherein the amino acid comprises proline.

[0169] 49. The method according to any one of the embodiments, wherein the enzyme comprises a pyrroline-5-carboxylate synthase (P5CS) molecule.

[0170] 50. The method according to any one of the embodiments, wherein the heterogeneous nucleic acid sequence includes a sequence encoding a P5CS molecule.

[0171] 51. The method according to any one of Embodiments 5 to 50, wherein the inhibitor is an enzyme substrate, or an analog, variant, or derivative thereof.

[0172] 52. The method according to any one of the embodiments, wherein the inhibitor is an analog of proline, such as L-azetidine-2-carboxylic acid, 3,4-dehydro-L-proline, or L-4-thiazolidinedic acid.

[0173] 53. The method according to any one of Embodiments 5 to 52, wherein the amino acid comprises proline, and the enzyme and inhibitor are selected from Table 1.

[0174] 54. The method according to any one of Embodiments 5-16, 34, or 38-47, wherein the amino acid comprises tyrosine, and the enzyme and / or inhibitor is selected from Table 1.

[0175] 55. The method according to any one of Embodiments 5-16, 34, 38-47, or 54, wherein the heterogeneous nucleic acid sequence comprises a sequence encoding an inhibitor of polypeptides and / or tyrosine biosynthesis enzymes.

[0176] 56. The method according to Embodiment 54 or 55, wherein the inhibitor is an analog of the enzyme substrate.

[0177] 57. The method according to any one of embodiments 5-16, 34, 38-47 or 54-56, wherein the inhibitor is a tyrosine analog.

[0178] 58. The method according to any one of Embodiments 5-16, 34, 38-47, or 54-57, wherein the amino acid comprises tyrosine, and the enzyme and inhibitor are selected from Table 1.

[0179] 59. The method according to any one of Embodiments 5-16, 34, or 38-47, wherein the amino acid comprises tryptophan and the enzyme and / or inhibitor is selected from Table 1.

[0180] 60. The method according to any one of Embodiments 5-16, 34, 38-47, or 59, wherein the heterogeneous nucleic acid sequence comprises a sequence encoding an inhibitor of polypeptides and / or tryptophan biosynthesis enzymes.

[0181] 61. The method according to Embodiment 59 or 60, wherein the inhibitor is an analog of the enzyme substrate.

[0182] 62. The method according to any one of embodiments 5-16, 34, 38-47 or 59-61, wherein the inhibitor is an analog of tryptophan.

[0183] 63. The method according to any one of Embodiments 5-16, 34, 38-47, or 59-62, wherein the amino acid comprises tryptophan, and the enzyme and inhibitor are selected from Table 1.

[0184] 64. The method according to any one of the embodiments, comprising the step of culturing cells that do not have high enzyme activity together with cells that have high activity.

[0185] 65. The method according to any one of the embodiments, wherein highly active cells grow more rapidly than non-highly active cells, for example, by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 times faster.

[0186] 66. The method according to any one of the embodiments, wherein less than about 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 percent of selected cells, for example based on growth, lack nucleic acids.

[0187] 67. A method according to any one of the embodiments, further comprising the step of selecting cells that show growth.

[0188] 68. The method according to any one of the embodiments, wherein the cells include eukaryotic cells.

[0189] 69. The method according to any one of the embodiments, wherein the cells include animal cells.

[0190] 70. The method according to any one of the embodiments, wherein the cells include mammalian cells.

[0191] 71. The method according to any one of the embodiments, wherein the cells include rodent cells.

[0192] 72. The method according to any one of the embodiments, wherein the cells include CHO cells.

[0193] 73. The method according to any one of the embodiments, wherein the cells include GSKO CHO cells.

[0194] 74. The method according to any one of the embodiments, wherein an endogenous copy of the enzyme-coding sequence is inactivated, for example, by deletion of a structural or regulatory region.

[0195] 75. The method according to any one of the embodiments, wherein an endogenous copy of the sequence encoding a second amino acid synthase is inactivated, for example, by deletion of a structural or regulatory region.

[0196] The method according to any one of the embodiments, wherein an endogenous copy of the sequence encoding 76.GS is inactivated, for example, by a deletion of a structural or regulatory region.

[0197] 77. A method according to any one of the embodiments, wherein the target nucleic acid sequence encodes a peptide molecule.

[0198] 78. The method according to any one of the embodiments, wherein the target nucleic acid sequence is heterogeneous.

[0199] 79. The method according to any one of the embodiments, wherein a different peptide molecule is selected from any of Tables 5 to 8.

[0200] 80. The method according to any one of the above embodiments, comprising the step of selecting cells to grow in a culture medium.

[0201] 81. The method according to embodiment 80, wherein the medium contains an inhibitor.

[0202] 82. 1) A step of selecting cells growing in a medium, 2) A step of culturing the cells, for example, subjecting the selected cells to a second selection, under a second set of culture conditions, for example, in a second medium. The method according to any one of the above embodiments, comprising:

[0203] 83. 1) The medium in 1) contains an inhibitor, 2) The second medium in 2) contains an inhibitor. The method according to embodiment 82.

[0204] 84. 1) A step of selecting cells growing in a medium, 2) A step of culturing the cells, for example, subjecting the selected cells to a second selection, under a second set of culture conditions, for example, in a second medium. The method according to any one of the above embodiments, comprising: wherein the concentration of the inhibitor in one of 1) and 2) is greater than the concentration of the inhibitor in the other of 1) and 2).

[0205] 85. The method according to embodiment 84, wherein the concentration of the inhibitor in 1) is greater than the concentration of the inhibitor in 2).

[0206] 86. The method according to embodiment 84, wherein the concentration of the inhibitor in 1) is greater than the concentration of the inhibitor in 2).

[0207] 87. The method according to any one of embodiments 84 to 86, wherein the medium in the step having a lower concentration of the inhibitor is essentially free of the inhibitor.

[0208] 88. The method according to any one of the above embodiments, wherein the nucleic acid sequence in (ii) is operably linked to a control sequence, such as a promoter.

[0209] 89. The method according to Embodiment 88, wherein the control sequence includes a sequence selected from SV40, mCMV, hCMV, or PGK promoter or a variant thereof.

[0210] 90. The method according to Embodiment 88, wherein the regulatory sequence controls the expression of LMM.

[0211] 91. The method according to Embodiment 90, wherein the control array and LMM are as listed in any single row of Table 3.

[0212] 92. The method according to Embodiment 90, wherein the control sequence comprises the SV40 promoter and the LMM is mouse SCD1.

[0213] 93. The method according to Embodiment 90, wherein the control sequence includes an SV40 promoter and the LMM is SCD1, for example, CHO SCD1.

[0214] 94. The method according to Embodiment 90, wherein the control sequence includes an SV40 promoter and the LMM is SREBF1, for example, CHO SREBF1.

[0215] 95. The method according to Embodiment 90, wherein the control sequence includes an SV40 promoter and the LMM is SREB411, for example, CHO SREB411.

[0216] 96. The method according to Embodiment 90, wherein the control sequence comprises an mCMV promoter and the LMM is mouse SCD1.

[0217] 97. The method according to Embodiment 90, wherein the control sequence comprises an mCMV promoter and the LMM is SCD1, for example, CHO SCD1.

[0218] 98. The method according to Embodiment 90, wherein the control sequence comprises an mCMV promoter and the LMM is SREBF1, for example, CHO SREBF1.

[0219] 99. The method according to embodiment 90, wherein the control array comprises the mCMV promoter and the LMM is SREB411, such as CHO SREB411.

[0220] 100. The method according to embodiment 90, wherein the control array comprises the hCMV promoter and the LMM is mouse SCD1.

[0221] 101. The method according to embodiment 90, wherein the control array comprises the hCMV promoter and the LMM is SCD1, such as CHO SCD1.

[0222] 102. The method according to embodiment 90, wherein the control array comprises the hCMV promoter and the LMM is SREBF1, such as CHO SREBF1.

[0223] 103. The method according to embodiment 90, wherein the control array comprises the hCMV promoter and the LMM is SREB411, such as CHO SREB411.

[0224] 104. The method according to embodiment 90, wherein the control array comprises the PGK promoter and the LMM is mouse SCD1. <​​​​​​​​​​​​​​​​​​109. The method according to Embodiment 108, wherein the control sequence, LMM and inhibitor (e.g., P5CS inhibitor) are as listed in any single row of Table 4.

[0230] 110. The method according to any one of Embodiments 88 to 109, wherein the culture medium contains L-azetidine-2-carboxylic acid.

[0231] 111. The method according to any one of embodiments 88 to 110, wherein the culture medium comprises 3,4-dehydro-L-proline.

[0232] 112. The method according to any one of Embodiments 88 to 111, wherein the culture medium contains L-4-thiazolidinedic acid.

[0233] 113. A method according to any one of the embodiments, further comprising the step of recovering the product of the target nucleic acid sequence.

[0234] 114. A method according to any one of the embodiments, comprising the step of recovering the product from the culture medium.

[0235] 115. A method according to any one of the embodiments, comprising the step of recovering the product from cells.

[0236] 116.i) A step of selecting cells to grow in the culture medium, ii) The step of culturing cells in a second medium, for example, under a second set of culture conditions, or subjecting selected cells to a second selection, iii) A step of recovering the product from the cells or a second culture medium. A method according to any one of the above embodiments, including the above.

[0237] 117. A method for identifying, selecting, or culturing cells containing a target nucleic acid sequence, a)(i) Target nucleic acid sequence and (ii) When expressed, nucleic acid sequences that result in high levels of activity of enzymes in amino acid synthesis pathways, such as the proline synthesis pathway, for example, nucleic acid sequences that encode enzyme molecules containing activity The steps include: preparing cells containing nucleic acids, such as vectors, such as replicable vectors; b) A step of culturing cells containing nucleic acid sequences in a first medium (and optionally containing an enzyme inhibitor) having levels of amino acids, such as proline, that are insufficient to support the growth of cells that are the same as non-highly active target cells, under conditions sufficient to enable the growth of cells containing nucleic acid sequences, c) A step of culturing cells containing a nucleic acid sequence in the presence of a second medium (and optionally a second medium containing an inhibitor of the second enzyme) containing a second amino acid, such as tyrosine, at a level insufficient to support the growth of cells that are the same as the unactive target cells, under conditions sufficient to enable the growth of the nucleic acid sequence-containing cells. A method for identifying, selecting, or culturing cells containing heterogeneous nucleic acid sequences, including the above.

[0238] 118. The method according to embodiment 117, wherein step b is initiated before step c begins.

[0239] 119. The method according to embodiment 117 or 118, wherein step b is performed before step c.

[0240] 120. The method according to embodiment 117 or 118, wherein steps b and c are performed simultaneously.

[0241] 121. The method according to any one of Embodiments 117 to 120, wherein steps b and c are performed in the same culture medium.

[0242] 122. The method according to any one of embodiments 117 to 121, wherein steps b and c are performed in the same container.

[0243] 123. The selection in step b and the selection in step c are performed in the same medium, and the medium is (a) Having insufficient levels of amino acids, such as proline, to support the growth of cells that are the same as the target cells that do not have high activity, (b) The same cells as the target cells that do not have high activity of the second enzyme, which have a level of the second amino acid, e.g., tyrosine, that is insufficient to support cell growth. The method according to any one of embodiments 117 to 122.

[0244] 124. Cells, (iii) A nucleic acid sequence that, if expressed, results in a high level of activity of the second enzyme in the amino acid synthesis pathway, for example, a nucleic acid sequence that encodes an enzyme molecule containing activity. The method according to any one of embodiments 117 to 123, further including the method described above.

[0245] 125. The method according to any one of embodiments 117 to 124, wherein cells are cultured in a medium having an inhibitor of enzyme activity.

[0246] 126. The method according to any one of embodiments 117 to 125, wherein cells are cultured in a medium having an inhibitor of the activity of a second enzyme.

[0247] 127. The method according to any one of Embodiments 117 to 126, wherein the second enzyme is located in the same pathway as the enzyme in (ii), for example, in the proline synthesis pathway.

[0248] 128. The second enzyme is a nucleic acid sequence that encodes an active enzyme molecule, for example, present in the same amino acid synthesis pathway. The culture medium in b) iv) Inhibitors of the activity of the second enzyme Further including, The method according to Embodiment 127.

[0249] 129. The method according to any one of Embodiments 117 to 128, wherein the second enzyme is located in a different pathway from the enzyme in (ii), for example, the enzyme is located in the proline synthesis pathway and the second enzyme is located in a pathway other than proline, for example, the tyrosine or tryptophan pathway.

[0250] 130. The second enzyme is present in the synthesis pathway of the second amino acid and is, for example, a nucleic acid sequence encoding an enzyme molecule containing activity. The culture medium in b) iii) A second amino acid, such as an amino acid other than proline, at a level insufficient to support the growth of the same cells as the target cells that do not have high activity, iv) Inhibitors of the activity of the second enzyme and Further including, The method described in Embodiment 129.

[0251] 131. Cells containing a sequence that includes a regulatory region from any of the following: SV40 promoter sequence, mCMV promoter sequence, or PGK promoter sequence, for example, a sequence that encodes a heterologous lipid metabolism modifier (LMM) operably linked to the promoter sequence.

[0252] 132. Cells according to Embodiment 131, wherein LMM modifies the pathways listed in Table 2.

[0253] 133. The cell according to Embodiment 131 or 132, wherein LMM contains sterol-CoA desaturase-1 (SCD1).

[0254] 134. Cells according to Embodiment 131 or 132, wherein LMM contains sterol regulatory element-binding transcription factor 1 (SREBF1).

[0255] 135. The cell according to Embodiment 131 or 132, wherein LMM comprises a cleaved isoform of SREBF1 (e.g., a cleaved isoform of SREBF1 lacking a regulatory domain, e.g., SREB411).

[0256] 136. A cell according to any one of the embodiments, wherein a sequence encoding a heterologous lipid metabolism modifier (LMM) is arranged in the vector.

[0257] 137. The cell according to any one of the embodiments, wherein the vector further comprises a sequence encoding a selectable marker, for example, a marker that, if present, enables survival or growth in a specified medium.

[0258] 138. The cell according to any one of the embodiments, further comprising a sequence encoding a selectable marker that, if present, enables survival or growth in a medium lacking nutrients, such as amino acids.

[0259] 139. The cell according to Embodiment 138, wherein the nutrient is an amino acid, for example, an amino acid selected from the amino acids listed in Table 1.

[0260] 140. A cell according to Embodiment 139, wherein the nutrient contains proline.

[0261] 141. The method according to any one of Embodiments 137 to 140, wherein the marker comprises a pyrroline-5-carboxylic acid synthase (P5CS) molecule.

[0262] 142. A cell according to any one of embodiments 138 to 141, wherein the nutrient contains tyrosine.

[0263] 143. A cell according to any one of embodiments 138 to 142, wherein the nutrient contains tryptophan.

[0264] 144. Cells according to any one of Embodiments 131 to 143, wherein the marker is selected from Table 1.

[0265] 145. A cell according to any one of embodiments 137 to 144, wherein the marker contains glutamine synthetase.

[0266] 146. A cell according to any one of embodiments 131 to 145, wherein the sequence encoding LMM is operably linked to the sequence encoding the SV40 promoter sequence.

[0267] 147. A cell according to any one of embodiments 131 to 145, wherein the sequence encoding LMM is operably ligated to the sequence encoding the mCMV promoter sequence.

[0268] 148. A cell according to any one of embodiments 131 to 145, wherein the sequence encoding LMM is operably linked to the sequence encoding the PGK promoter sequence.

[0269] 149. A cell according to any one of embodiments 131 to 148, wherein LMM is SCD1.

[0270] 150. The cell according to Embodiment 149, wherein LMM is mouse SCD1.

[0271] 151. The cell according to Embodiment 149, wherein LMM is CHO SCD1.

[0272] 152. A cell according to any one of embodiments 131 to 148, wherein LMM is SREBF1.

[0273] 153. A cell according to any one of embodiments 131 to 148, wherein LMM is CHO SREBF1.

[0274] 154. A cell according to any one of embodiments 131 to 148, wherein LMM is SREB411.

[0275] 155. A cell according to any one of embodiments 131 to 148, wherein LMM is CHO SREB411.

[0276] 156. A cell according to any one of Embodiments 131 to 148, wherein LMM contains sterol-CoA desaturase-1 (SCD1) and the promoter contains the SV40 promoter sequence.

[0277] 157. A cell according to any one of Embodiments 131 to 148, wherein LMM contains sterol-CoA desaturase-1 (SCD1) and the promoter contains an mCMV promoter sequence.

[0278] 158. A cell according to any one of Embodiments 131 to 148, wherein LMM contains sterol-CoA desaturase-1 (SCD1) and the promoter contains a PGK promoter sequence.

[0279] 159. A cell according to any one of Embodiments 131 to 148, wherein LMM contains sterol regulatory element-binding transcription factor 1 (SREBF1) and the promoter contains the SV40 promoter sequence.

[0280] 160. A cell according to any one of Embodiments 131 to 148, wherein LMM contains sterol regulatory element-binding transcription factor 1 (SREBF1) and the promoter contains an mCMV promoter sequence.

[0281] 161. A cell according to any one of Embodiments 131 to 148, wherein LMM contains sterol regulatory element-binding transcription factor 1 (SREBF1) and the promoter contains a PGK promoter sequence.

[0282] 162. A cell according to any one of Embodiments 131 to 148, wherein LMM comprises SREB411 and the promoter comprises the SV40 promoter sequence.

[0283] 163. A cell according to any one of Embodiments 131 to 148, wherein LMM comprises SREB411 and the promoter comprises an mCMV promoter sequence.

[0284] 164. A cell according to any one of Embodiments 131 to 148, wherein LMM contains the sterol SREB411 and the promoter contains a PGK promoter sequence.

[0285] 165. Cells according to any one of Embodiments 131 to 148, wherein the culture medium contains the inhibitors listed in Table 1 or 4.

[0286] 166. Cells according to any one of embodiments 131 to 148, wherein the culture medium contains a P5CS inhibitor.

[0287] 167. The cell according to Embodiment 166, wherein the P5CS inhibitor is L-azetidine-2-carboxylic acid.

[0288] 168. The cell according to Embodiment 166, wherein the P5CS inhibitor is 3,4-dehydro-L-proline.

[0289] 169. The cell according to Embodiment 166, wherein the P5CS inhibitor is L-4-thiazolidinediocarboxylic acid. [Examples]

[0290] Example 1: Investigation of inhibitors for use in mammalian selective systems for pyrroline-5-carboxylic acid synthase expression and proline metabolism. Pyrroline-5-carboxylic acid synthase (P5CS) is the rate-limiting enzyme responsible for proline synthesis from glutamate. A simplified diagram of this pathway is shown in Figure 1. Since proline metabolism is essential for cell survival, cells cultured in proline-free medium should not be able to survive. Alternatively, cells with abundant P5CS expression and therefore the ability to efficiently produce proline from glutamate should enable cell survival under these conditions and thus facilitate the appropriate selection of desired cells.

[0291] To evaluate this hypothesis, a vector containing the P5CS gene was first synthesized and cloned to produce a construct containing both the target gene and the eGFP reporter gene (Figure 2). Upon transfection, this vector should induce P5CS expression levels exceeding those endogenously observed in CHO cells, enabling high-rate proline synthesis and thus promoting cell survival in the absence of extraproline normally present in the culture medium. Theoretically, these cells expressing abundant P5CS for transfection of the construct should also express the eGFP gene.

[0292] Overexpression of P5CS may be sufficient for cell selection in the absence of proline, but inhibitors of this enzyme can improve the rigor of this system. Since proline is part of the endogenous feedback loop in CHO cells, proline itself is an effective inhibitor of P5CS. Inhibitors specified for use in this system include analogues of proline. Exemplary inhibitors that are effective inhibitors of the in vitro activity of P5CS are shown herein.

[0293] Cell growth in the absence of proline In one experiment, cells engineered to knock out endogenous glutamine synthetase expression (GSKO cells) were cultured in the absence of proline to determine the effect of this on growth. GSKO cells were cultured in 125 ml Erlenmeyer flasks at a total culture volume of 20 ml, resulting in 0.2 x 10⁴ cells. 6 Cells were seeded at a concentration of 100 cells / ml. The cells were cultured in CD-CHO with or without L-glutamine (6 mM Glut), or in CM76 medium with 6 mM L-glutamine but without proline (Pro-less). Live cell counts and culture viability were measured every 24 hours using a ViCell instrument.

[0294] GSKO cells were unable to grow when cultured in the absence of proline (Figure 3). However, cells cultured in the absence of L-glutamine showed a decrease in culture viability over time. This was expected, as these cells had been engineered to knock out the endogenous expression of glutamine synthetase, the rate-limiting enzyme in glutamine production.

[0295] Recovery rate of cells cultured in the absence of proline The number of revertant cells resulting from cultures incubated in proline-free medium was determined to evaluate the suitability of using proline metabolism in the selection system. Briefly, Lonza GSKO cells were seeded in either CD-CHO with 6 mM L-glutamine, CD-CHO without L-glutamine, or CM76 with 6 mM L-glutamine but without proline, at a rate of either 1,000 or 5,000 cells per well in a 96-well plate, with a culture volume of 200 μl per well. A total of 30 plates were seeded in each medium: 15 plates were cultured with 1,000 cells per well, and 15 plates were cultured with 5,000 cells per well. Revertant colonies were identified by microscopic observation after 11 days.

[0296] 100% of wells seeded in CD CHO with 6 mM L-glutamine were confluent after 11 days, but no revertant colonies were observed when cultured in L-glutamine-free medium. A total of 8.64 × 10⁶ colonies were observed when cultured in the absence of proline. 6 Of the individual cells, 15 revertant colonies were observed; 13 were observed on plates seeded with 5,000 cells per well, and 2 colonies were observed on plates seeded with 1,000 cells per well.

[0297] Overexpression of P5CS to select cells that express the target gene. As shown above, the removal of proline from the culture medium results in a loss of growth in GSKO cells. We hypothesized that overexpression of pyrroline-5-carboxylic acid synthase (P5CS), the rate-limiting enzyme in endogenous proline synthesis, would be sufficient to restore growth in the absence of exogenous proline, thereby providing an effective selection system. To evaluate this hypothesis, cells were transfected with plasmids containing the P5CS gene to promote its overexpression and cultured in the absence of proline. Briefly, the plasmids were constructed to contain either glutamine synthetase (GS) or P5CS, and the eGFP reporter gene. A control vector without any "selection" gene or reporter was also included; the control vector contained only the etanercept gene to induce a similar cellular load as the other plasmids involved in this experiment. A schematic diagram of the vectors used is shown in Figure 4. The constructed plasmid was transiently transfected into GSKO cells by electroporation and cultured in various media: CD-CHO, tyrosine-free CM76 medium, or proline-free CM76 medium (all of which were supplemented to a final concentration of 6 mM L-glutamine). The total culture volume in a 125 ml Erlenmeyer flask, shaken at 140 rpm at 37°C, was 20 ml (originally 0.2 x 10⁻⁶). 6 Samples were collected for counting and analysis by flow cytometry at 72 and 168 hours post-transfection (seeded at 100 cells / ml).

[0298] Figure 5 shows data obtained after transfection of GSKO cells transiently transfected with the vector outlined in Figure 4 and then cultured in specific media. Cells cultured in either tyrosine-free or proline-free media showed reduced growth rate at 72 hours post-transfection compared to cells cultured in CD-CHO, regardless of which plasmid was introduced (Figure 5A). This observation is consistent with the pattern outlined above. However, at 168 hours post-transfection with the P5CS-containing vector, an increase in viable cell concentration was observed in proline-free medium compared to when any other vector was used. Furthermore, the introduction of this vector did not result in the increase in viable cell count achieved in tyrosine-free medium, suggesting that P5CS can compensate, in particular, for the absence of exogenous proline.

[0299] Despite observable differences in viable cell concentrations, culture viability was generally not significantly affected by culture in different media (Figure 5B). It is noteworthy that transfection with the P5CS-containing construct, and subsequent 72-hour culture, yielded lower values ​​than those obtained from other transfections cultured in the same medium. This may be due to the additional cellular load imposed on these cells due to increased proline synthesis in addition to the deficiency of available tyrosine. This trend was not observed at 168 hours. While cell viability was generally lower at 168 hours in CD-CHO, this appears to be due to the typical growth profile of these cells, which are expected to be in a decline phase at this point, in contrast to cells cultured in other media that did not follow a typical growth trend.

[0300] Flow cytometry analysis showed that the combination of transfection with a P5CS-containing plasmid followed by culture in proline-free medium resulted in the highest fluorescence mean values ​​among the conditions measured at both time points (Figure 5C). In general, cells cultured in the absence of proline showed higher fluorescence mean values ​​than cells cultured without exogenous tyrosine, and the inclusion of P5CS in a transient plasmid followed by culture in tyrosine-free medium showed higher fluorescence mean values ​​than when GS was included instead, suggesting that P5CS overexpression may have a broad effect on increasing eGFP expression. However, the combination of P5CS overexpression was clearly more effective on cells subsequently cultured in proline-free medium, as confirmed by a large increase in fluorescence mean values ​​and reinforced by the significantly increased values ​​observed in the percentage of cells exceeding a given fluorescence intensity threshold (Figure 5D).

[0301] These data, when combined, support the hypothesis that the suppressed cell growth observed when GSKO cells are cultured in the absence of proline can be restored through overexpression of the P5CS gene. Furthermore, this may have a significant effect on reporter gene expression, both in terms of overall expression and the percentage of cells expressing the reporter gene within a transient population. Generation of a stable cell pool that expresses the target gene. To assess the ability to generate a recombinant cell pool of the proline / P5CS system, a vector containing the P5CS gene and the target gene was transfected into cells in an attempt to generate a recombinant cell pool, and then cultured in the absence of proline. Briefly, a 20 μg plasmid, first linearized using PvuI restriction enzyme (NEB) and purified using sodium acetate and ethanol precipitation, was then transfected into 1 x 10⁶ cells. 7GSKO cells from Lonza were electroporated. The construct used contained both the P5CS gene for evaluation as a selection marker and eGFP as a reporter gene. Transfection was performed in CM76 medium supplemented with L-glutamine to achieve a final concentration of 6 mM, but in the absence of proline. The cells were then cultured in 25 ml of this medium in a T75 flask for 10 days, and then transferred to a 10 ml suspension culture in proline-free medium. The resulting pool was analyzed using flow cytometry. Cell samples were first centrifuged at 1,000 rpm for 5 minutes and resuspended in 500 μl of PBS. The samples were then loaded onto a FACScalibur (BD biosciences) probe, and fluorescence intensity was measured in relation to the cell count. Using an E-1 amplifier, forward scatter (FSC) was measured, and side scatter (SSC) was set to 465, while FL1 recording cells were set to 473; all settings were converted to a log scale. To promote adhesion of the suspended cells, the coverslips were first immersed in poly-L-lysine and incubated at room temperature for 15 minutes. The coverslips were then removed, dried in a sterile environment, and then transferred to a 24-well plate. The cells were cultured overnight at 37°C to promote adhesion to the coverslips, then the medium was aspirated, 4% paraformaldehyde in 250 μl of PBS was added, and the cells were fixed by incubation at 37°C for 20 minutes. Next, the cells were permeabilized by two 1 ml PBS washes, then 0.1% Triton X100 in 250 μl of PBS was added, and the cells were incubated at room temperature for 5 minutes. The fixed and permeabilized cells were sealed by adding 3% BSA in 250 μl of PBS per well and incubated at room temperature for 30 minutes. This was aspirated, the coverslip was removed from the well, and placed face down on a previously prepared 100 μl PBS droplet. This was repeated three more times. The coverslip was dried by touching its edge to a tissue paper and transferred to a 25 μl droplet of appropriate primary antibody. This was incubated overnight at 4°C. Appropriate secondary antibodies were diluted as needed in 3% BSA in PBS.The cover glass was placed upside down on a 0.1% Tween droplet in 100 μl of PBS and left for 5 minutes. Then, these were transferred to fresh droplets four times, leaving them for 10 minutes between the last two transfers. Then, they were placed on 25 μl of secondary antibody droplets, left as such for 2 hours, and placed in a dark room at 4°C. Five sequential 100 μl droplets were used for five final washes. If DAPI staining was required, the cover glasses were each transferred to a 50 μl droplet of 10 mg / ml DAPI between the second and third PBS droplets. Finally, the cells were mounted on cell slides using ProLong Gold anti-fade mountant (ThermoScientific) and left overnight to fix. Images were collected using a Zeiss confocal microscope. The host cells were 10. 1 Calibrate the flow cytometer instrument so that the fluorescence intensity values do not exceed a certain threshold. Therefore, cells exceeding this predetermined threshold are considered to express eGFP.

[0302] Figure 6A shows a histogram obtained using flow cytometry from a pool generated using the P5CS-based selection system. Many cells have a fluorescence intensity exceeding 10 1 Since the threshold, it is clear that the P5CS selection system can select cells expressing the gene of interest. However, there is a population of cells with fluorescence intensity values less than 10 1 indicating that non-expressing cells are still present in the pool. It may be important to increase the stringency of the system. Figure 6B shows an image of the synthesized pool obtained using a fluorescence microscope. There are populations of cells with various eGFP expression levels, and not all cells present necessarily express the eGFP reporter gene, so this image supports the data shown in Figure 6A.

[0303] Both Figures 6B and 6C show the expression patterns of the P5CS gene. These images show punctate spots consistent with the localization of P5CS in mitochondria previously reported in the literature. Furthermore, Figure 6C shows a comparison of the P5CS / eGFP pool with the GSKO host. The image shows that the selected pool has higher P5CS expression levels than the host, although this observation is not exclusive to cells expressing the eGFP reporter gene. Since a subset of host cells has been shown to undergo reversion in the absence of proline, it is possible that cells not containing the transfected vector were able to survive the selection process outlined without requiring overexpression of P5CS. Also, perhaps the absence of proline selects cells that endogenously express sufficient P5CS.

[0304] Cell pools constructed using the P5CS system were examined by Western blotting of solubilized samples taken from the cell pools. All vectors used to generate these pools contained P5CS and one of the following genes as labeled: eGFP or SCD1 (driven by either the SV40 or mCMV promoter). Control samples were constructed using a vector containing only P5CS. GSKO cells were transfected with the linearized vectors mentioned above by electroporation, cultured for two weeks in the absence of proline, and then transferred to suspension culture. As shown in Figure 7, Western blotting of solubilized hydrates from the resulting cell pools showed that SCD1 was overexpressed in the relevant cell pools (SV40 SCD1 and mCMV SCD1) compared to eGFP and the blank control, indicating that the P5CS system is suitable for the cell line construction process. In addition, we also transfected cells with a control vector lacking the P5CS gene, but the resulting cells did not grow in the absence of proline, demonstrating that P5CS overexpression is extremely important for cell survival.

[0305] Cell pool grown in the presence of a P5CS inhibitor Cell pool grown in a deep 96-well plate Data were obtained from a cell pool constructed using the P5CS / eGFP vector (as outlined above) when cultured in a medium lacking proline but supplemented with various concentrations of the P5CS inhibitor L-azetidine-2-carboxylic acid. Cells were cultured in deep 96-well plates and 0.2 x 10⁶ wells. 6 Cells were seeded in a suspension at a rate of 10 cells / ml. Number of viable cells (Figure 8A), culture viability (Figure 8B), average fluorescence value (Figure 8C), 10 2 Cells expressing above a predetermined fluorescence threshold (Figure 8D) and 10 3 Cells expressing eGFP above a predetermined fluorescence threshold (Figure 8E) were identified at 24, 96, 168, and 216 culture times. Increasing the inhibitor concentration resulted in slower cell growth, but higher fluorescence mean values ​​were also observed, indicating that the addition of the inhibitor can enrich eGFP expression from the existing polyclonal population. Figure 8F shows Western blot analysis from solubilized samples collected at the same time point. The blots were probed to highlight P5CS, β-actin, and eGFP.

[0306] Data were obtained from a cell pool constructed using the P5CS / eGFP vector (as outlined above) when cultured in a medium lacking proline but supplemented with various concentrations of the P5CS inhibitor 3,4-dehydro-L-proline. Cells were cultured in deep 96-well plates and measured 0.2 x 10⁶ cells. 6 Cells were seeded in a suspension at a rate of 10 cells / ml. Number of viable cells (Figure 9A), culture viability (Figure 9B), average fluorescence value (Figure 9C), 10 2 Cells expressing above a predetermined fluorescence threshold (Figure 9D) and 10 3 All cells expressing eGFP above a predetermined fluorescence threshold (Figure 9E) were identified at 24, 96, 168, and 216 culture times. Increasing the inhibitor concentration resulted in slower cell growth, but higher fluorescence mean values ​​were also observed, indicating that the addition of the inhibitor can enrich eGFP expression from the existing polyclonal population. Figure 9F shows Western blot analysis from solubilized samples collected at the same time point. The blots were probed to highlight P5CS, β-actin, and eGFP.

[0307] Data were obtained from a cell pool constructed using the P5CS / eGFP vector (as outlined above) when cultured in a medium lacking proline but supplemented with various concentrations of the P5CS inhibitor L-4-thiazolidinecarboxylic acid. Cells were cultured in deep 96-well plates and 0.2 x 10⁶ wells. 6 Cells were seeded in suspension at a rate of 10 cells / ml. Number of viable cells (Figure 10A), culture viability (Figure 10B), average fluorescence value (Figure 10C), 10 2 Cells expressing above a predetermined fluorescence threshold (Figure 10D) and 10 3 All cells expressing eGFP above a predetermined fluorescence threshold (Figure 10E) were identified at 24, 96, 168, and 216 culture times. Increasing the inhibitor concentration resulted in slower cell growth, but higher fluorescence mean values ​​were also observed, indicating that the addition of the inhibitor can enrich eGFP expression from the existing polyclonal population. Figure 10F shows Western blot analysis from solubilized samples collected at the same time point. The blots were probed to highlight P5CS, β-actin, and eGFP.

[0308] Cell pool grown in a 24-well plate Data were obtained from a cell pool constructed using the P5CS / eGFP vector (as outlined above) when cultured in a medium lacking proline but supplemented with various concentrations of the P5CS inhibitor L-azetidine-2-carboxylic acid. Cells were cultured in fixed 24-well plates, initially at 0.2 x 10⁶ 6 Cells were seeded at a rate of 10 cells / ml. Number of viable cells (Figure 11A), culture viability (Figure 11B), average fluorescence value (Figure 11C), 10 2 Cells expressing above a predetermined fluorescence threshold (Figure 11D) and 10 3 All cells expressing eGFP above a predetermined fluorescence threshold (Figure 11E) were identified at 24, 96, 168, and 216 culture times. Increasing the inhibitor concentration resulted in slower cell growth, but higher fluorescence mean values ​​were also observed, indicating that the addition of the inhibitor can enrich eGFP expression from the existing polyclonal population.

[0309] Data were obtained from a cell pool constructed using the P5CS / eGFP vector (as outlined above) when cultured in a medium lacking proline but supplemented with various concentrations of the P5CS inhibitor 3,4-dehydro-L-proline. Cells were cultured in fixed 24-well plates, initially at 0.2 x 10⁶ 6 Cells were seeded at a rate of 10 cells / ml. Number of viable cells (Figure 12A), culture viability (Figure 12B), average fluorescence value (Figure 12C), 10 2 Cells expressing above a predetermined fluorescence threshold (Figure 12D) and 10 3 All cells expressing eGFP above a predetermined fluorescence threshold (Figure 12E) were identified at 24, 96, 168, and 216 culture times. Increasing the inhibitor concentration resulted in slower cell growth, but higher fluorescence mean values ​​were also observed, indicating that the addition of the inhibitor can enrich eGFP expression from the existing polyclonal population.

[0310] Example 2: Lipid metabolism modifiers for improving host cell antibody production Manipulation of SREB411, a cleaved SREBF1 isoform containing the lipid metabolism modifier (LMM) stearoyl-CoA desaturase-1 (SCD1), sterol regulatory element-binding transcription factor 1 (SREBF1), and a nuclear-migrating portion of SREBF1 lacking the regulatory domain of SREBF1, has been shown to improve CHOK1SV GS-KO host production and the product quality of the complex protein and standard mAb.

[0311] It was hypothesized that if the expression of these LMMs were modified in the CHOK1SV GS-KO host in the manner of current industrial cGMP, it could result in a significant improvement in product titer and quality. The objective was to insert a desired LMM gene into an existing cell line, or to create a new host cell line that expresses one of the LMM genes while keeping the commercially available GS selection system intact.

[0312] To achieve this objective and test the hypothesis, it was necessary to determine the expression levels and which of the LMM genes was most likely to be applicable. To achieve this objective, several vectors were produced: four vectors (A-D) expressing recombinant proteins and GS selection markers, and ten vectors (1-10) expressing specific promoter + LMM gene combinations (Table 9).

[0313] [Table 9]

[0314] Subsequently, all combinations of these vectors can be screened by co-transfecting one recombinant molecular vector with one promoter + LMM gene vector. Transfection was performed using a 4D-nucleofector (Nulcofector) (Lonza) and subsequent standard protocols; this can produce 96 repeat cultures per condition (co-transfection). The repeat pool of these transfected cells can then be assessed for cell growth during culture and recombinant protein production under fed batch conditions. These data can then be analyzed to determine which promoter + LMM combinations are suitable candidates for further analysis and / or cell line construction. GSKO cell growth and transfection GS-KO CHO cells were cultured in CD-CHO + 6 mM L-glutamine. The cultures were subcultured every 4 days; each 0.6 ml parent culture was counted using a Visell-XR instrument, and 50 images were recorded per culture. Daughter cultures were 0.2 x 10 6 The cells were seeded at a rate of 100 cells / ml; then, 5% CO2 was injected into the culture and incubated at 36.5°C and 140 rpm.

[0315] On the second or third day of passage, count the GS-KO cells using Viscell-XR; then count 2 x 10⁻¹⁰ cells. 7A volume equal to 10¹ viable cells / ml was collected and spun at 100g for 10 minutes. After centrifugation, the supernatant was discarded by pipetting, and the pellet was resuspended in 200 μl of SF solution (provided in the 4D-Nucleofector kit). The desired vector from transfection was then added to the 200 μl cell / SF suspension in a volume giving 6 μg per vector (LMM vector and recombinant protein). The DNA / cell / SF suspension was then equally divided in half and pipetted into 4D nucleofector cuvettes, taking care not to induce any foam formation in the cuvettes. The volume added to the cuvettes depended on the total volume after the addition of the DNA vector (typically between 10³ and 10⁸ μl).

[0316] The cuvette was then placed in a 4D nucleoeffector and pulsed using pulse code DU158. If both pulses failed (indicated by a red "-" on the display), a new 2×10 7 The volume of individual cells was collected, and the method was restarted. If a pulse passed (indicated by a green "+" on the display), approximately 500 μl of 40 ml of pre-warmed (37°C) transfection medium (40 ml CD-CHO, 0.4 ml SP4, 0.04 ml concentrated (x1000) phenol red) was pipetteed into the cuvette, and then the entire volume of culture was transferred to 40 ml of transfection medium; then, using a Pasteur pipette, any residual cells were "flushed" from the cuvette with transfection medium. The transfectant cell culture was then transferred to two 96-well plates (100 μl of culture per well) and incubated overnight at 36.5°C, 10% CO2.

[0317] On day 1 post-transfection, 100 μl of transfection medium + 100 μM MSX per well was added to each plate of transfected cultures. The plates / cultures were then incubated at 36.5°C in 10% CO2 for 6 days. On day 7 post-transfection, 150 μl of transfected culture per well was carefully removed from each plate of transfected cultures, and fresh transfection medium + 50 μM MSX was added to each well / culture. The cultures were then monitored for post-transfection recovery.

[0318] Approximately 10 days after transfection, we observed the cell recovery after transfection. This was done by visually inspecting the color of the transfection medium to assess the confluence of each well, and by using a cloning mirror. The bottom of wells where cells had grown may appear opaque, and the phenol red in the transfection medium may turn yellow upon lactic acid production; these were used as indicators of cell growth. When more than 75% of the culture was considered confluent (75-80%), the culture in the plate was considered recovered and transferred to a deep-well plate.

[0319] As shown in Figure 13, the recovery rates between recombinant proteins appeared to be similar, with the exception of cergutuzumab. This may be due to the structural properties of cergutuzumab; cergutuzumab has an IL-2 (interleukin-2) molecule bound to the Fc region of its antibody structure. This IL-2 may inhibit the growth rate of transfected cells (perhaps through its molecular action on cells after the cergutuzumab molecule is secreted from the cells). Alternatively, it may be because the mAb-IL-2 construction limits cellular resources, which slows the recovery rate.

[0320] Growth of transfected cell pools in deep well plates Once the culture had recovered, each well was mixed by pipetting to suspend the cells that had settled at the bottom. After suspending the cells in the culture, 150 μl was transferred to 150 μl of fresh medium (CD CHO, 1% SP4, 50 μM MSX) in a DWP. The deep-well plate containing the cell culture was then incubated at 36.5°C, 5% CO2, 200 rpm, and 90% humidity.

[0321] The first DWP subculturing was performed on either day 4, 5, or 6, depending on the growth rate of the culture. Cells were counted by Celigo; then, cells were divided into 0.5 x 10⁻⁶ cells. 6 Cells were seeded at a concentration of 1 / ml in 300 μl of CM66+ 50 μM MSX. Daughter DWPs were then incubated at 36.5°C, 5% CO2, 200 rpm, and 90% humidity. Subculturing was then performed on a 4-day schedule. Individual cultures / wells were counted using the Celigo system; the counts were then averaged across conditions to produce the results shown in Figure 14. In the first subculturing, the medium was changed from transfection medium to growth medium, and in the third subculturing, the growth medium was changed to production medium to initiate simple fed-batch overgrowth (aFOG).

[0322] Figures 14A–14D show the mean viable cell concentration (VCC) of all recovered transfected cells in deep-well plate cultures. Etanercept + mCMV SREB411 and etanercept + mCMV SREBF1 (Figure 14A) recovered at a slower rate than the other conditions. For practical reasons of passage the other conditions, it was decided to allow all conditions to enter aFOG at the same time by adding one extra passage. This was the reason for the extra passage recorded for the etanercept + LMM condition.

[0323] In general, the average VCC of cultures across conditions appeared to stabilize / equalize around the third passage, and the average VCC clustered together across all conditions.

[0324] Simple flow rate overgrowth of the transfectant pool To assess the productivity of the LMM cell pool, the pool was subjected to a flow-added overgrowth cycle to simulate bioreactor-scale conditions. Briefly, at the third passage in DWP, cells were counted using Celigo and then transferred to 300 μl of CM71 (0.3 x 10⁶ cells). 6 Cells were seeded at a concentration of 100 cells / ml. Cells were incubated at 36.5°C, 5% CO2, 200 rpm, and 90% humidity for 12 days. On days 4 and 8, cells were added to the culture and counted using Celigo. On day 12, the plate was centrifuged at 300 rpm for 10 minutes; 200 μl of supernatant was collected and placed in a sterile 96-well plate. The residual cell pellet supernatant was washed with 200 μl of PBS and centrifuged at 500 rpm for 10 minutes; then, as much of the PBS wash as possible (approximately 200 μl) was removed, and the cell pellet was transferred to -20°C. The collected supernatant was centrifuged at 3000 rpm, and then 180 μl was transferred to a new 96-well plate. The collected supernatant was then stored overnight at 4°C and analyzed the following day using an Octet instrument.

[0325] Cells are passed through at a lower cell density (0.3x10) than in routine passages. 6 VCC / ml:aFOG;0.5x10 6 VCC / ml (passaging) was used, and seeding was carried out in a production medium very similar to the subculturing growth medium; therefore, any change in the growth profile due to the change in medium should be minimal (Figures 15A-15D). VCC measurements were taken on the same day as feeding and harvesting; this allowed for a rough outline of the growth profile for each condition during production. This method provides limited growth data and therefore may miss growth peaks for some of the conditions. These missing data affect the calculation of IVC (time integral of viable cell concentration in culture per hour per milliliter) and Qp (specific productivity as pg / cells / hour); however, since all cultures and conditions have the same limitations, the data can be considered equivalent.

[0326] Some growth profiles under etanercept and infliximab conditions suggest that VCC peaks between days 4 and 8. SREBF1 expression may delay culture growth after day 4, as suggested by growth under the SV40 / PGK SREBF1 condition.

[0327] The variation at each time point is likely due to the fact that each counted culture is a pool of transfected cells; therefore, the copy numbers of both vectors fluctuate, which affects cell growth. We achieved mitigation of this variability by using 96 individual cultures per condition and taking the average. This suggests that if the combination of promoter, LMM gene, and recombinant protein has a significant effect, the overall variation in that condition may shift.

[0328] Measurement of product concentration after aFOG Product concentrations were measured using a protein A biosensor with Octet. Octet allows searching individual wells in a 96-well format, but two columns are required for plate standards and controls; therefore, a maximum of 80 samples could be measured per Octet run. Standards were prepared between 100 and 5 mg / L, inter-plate controls were diluted to 25 mg / L, and the recovered supernatant from each pool was diluted with CD-CHO to fit the resulting standard curve.

[0329] The sample requires a minimum concentration for the Octet sensor to quantify the product bound to the protein A biosensor. The number of quantifiable cultures varied between conditions; the total number of quantifiable cultures is listed in Table 10. These data show changes in the total number of quantifiable cultures across some LMM gene conditions in recombinant protein conditions; namely, SV40 SREB411 showed a decrease in quantifiable cultures across all recombinant protein conditions; the SV40 SREBF1 condition also showed a change, with a similar or improved number of quantifiable cultures.

[0330] [Table 10]

[0331] Since a pool of transfected cells was used, it was anticipated that there might be differences in the copy number of each transfected vector per cell in each pool, as well as in inherent productivity. To mitigate this variability and obtain a more accurate picture of cell growth and productivity under each condition, a maximum of 80 cell pools were produced per condition. The number of quantifiable replicates varied between 14 and 80 pools.

[0332] Calculation of specific productivity By collecting all protein A and growth data, it was possible to calculate specific productivity for each culture. This was done by calculating the pictogram per cell per hour (Qp) and the time integral of the viable cell concentration in the culture per hour per milliliter (IVC), with IVC being calculated by solving the area under the growth curve for each culture. These data could then be represented as Qp vs. IVC bubble plots, where each bubble (data point) indicates the product titer by its diameter. The resulting plots can be seen in Figures 16A-16D.

[0333] The 95% confidence intervals for the mean productivity data shown in Table 11 were analyzed for the mean values ​​of each RPLC (recombinant protein + promoter + LMM condition); these data are shown in Table 12, and the resulting intervals are shown in Table 13.

[0334] [Table 11]

[0335] [Table 12]

[0336] Subsequently, as shown in Figures 17A-17D, the data shown in Table 13 was plotted for each recombinant protein transfection.

[0337] Consideration By considering both specific productivity plots and 95% confidence interval plots (CIP), it was possible to identify promoter + LMM combinations that showed improved recombinant protein expression under these conditions.

[0338] Etanercept These data showed that, on average, the PGK+SREBF1 condition increased etanercept titer. This was observed from the average of 80 transfected cells.

[0339] serugutuzumab The SV40+SREB411 condition showed an increase in titer at 95% CIP.

[0340] Infliximab The number of measurable cultures for infliximab was generally lower compared to etanercept and cB72.3 conditions.

[0341] PGK / SV40+SREBF1 showed an increase in the titer of infliximab. In the infliximab bubble plot, the majority of data points with Qp=1 appeared to be from these conditions. The PGK+SREBF1 condition had a lower IVC than the SV40+SREBF1 condition, but the PGK+SREBF1 condition had a larger mean titer, which can also be seen by comparing the number of data points with Qp=1. Furthermore, these two conditions showed an increase in the number of measurable replicates, suggesting that SREBF1 expression at these levels increases cell viability when expressing infliximab.

[0342] cB72.3 The cB72.3 recombinant protein is a standard monoclonal antibody and was more readily expressed by cells compared to other recombinant proteins discussed herein.

[0343] 95% CIP suggested that increasing SREBF1 expression could increase the final titer under these conditions. Both SV40 / PGK+SREBF1 conditions showed a significant increase in titer. The PGK+SCD1 condition also showed an increase in titer. The cB72.3 bubble plot showed that the SV40+SREBF1 data tended towards higher Qp and lower IVC, while the opposite was true for the PGK+SREBF1 condition. The SCD1 condition appeared to be groupable between Qp=0.5–3 and IVC=500–1250. The control condition tended towards lower Qp and higher IVC.

[0344] Selection of Promoter and LMM Combinations The SV40 / PGK+SREBF1 combination showed improvement in product titer and either Qp or IVC of the transfected cultures. Differences between the mouse and CHO sequences for SCD1 can be explored with strong promoters such as hCMV or mCMV. Combinations of these promoters are intended to improve product titer.

[0345] Example 3: Application of a pyrroline-5-carboxylic acid synthase proline metabolic selectivity system for the generation of a GS-KO CHO cell pool stably introduced with LMM. Manipulation of SREB411, a cleaved SREBF1 isoform containing the lipid metabolism modifier (LMM) stearoyl-CoA desaturase-1 (SCD1), sterol regulatory element-binding transcription factor 1 (SREBF1), and a nuclear-migrating portion of SREBF1 lacking the regulatory domain of SREBF1, has been shown, as in the above example, to improve CHOK1SV GS-KO host production and the product quality of the complex protein and standard mAb. Therefore, P5CS proline metabolic selection was utilized to generate a stable expression LMM-modified GS-KO CHO cell pool overexpressing either SCD1 or SREBF1 under the control of various promoters.

[0346] Using a proline-deficient, Lonza-exclusive, known-composition protein-free medium, PGK SCD1(CHO) or PGK SREBF1, or PGK SCD1(mouse) or SV40 SREBF1, was transfected into a GS-KO CHO host by electroporation using a P5CS selection system. The transfected cells were then seeded into three 96-well plates (100 μl of culture per well) and incubated at 36.5°C and 10% CO2. Seven days post-transfection, 150 μl per well / culture was removed from each plate, and fresh proline-deficient medium was added to each well / culture. The cultures were then monitored for post-transfection growth and recovery. Post-transfection cell recovery was observed approximately 14 days post-transfection. If more than 75% of the culture was considered confluent (75-80%), the culture in the plate was considered recovered and transferred to a deep 96-well plate.

[0347] After subculturing in deep 96-well plates, cell pellet samples were collected, protein solubilizers were prepared, and the expression of the target LMM (SCD1 or SREBF1), along with P5CS expression and β-actin acting as a control, was analyzed by Western blotting. From the resulting analysis, the cell pools in each well were classified as expressing "high," "medium," or "low" levels of the target LMM compared to each other and to the control. Subsequently, the 10 most expressing cell wells were combined to create a "high" expression cell pool, 10-15 wells from the "medium" expression group were collected to create a "medium" expression LMM pool, and 10-15 wells from the "low" expression group were combined to create a "low" expression LMM pool. For the medium and low pools, wells were taken from each of the three plates. These LMM-introduced cell pools were then expanded into shaking flasks and grown in proline-free Lonza proprietary medium. Subsequently, the cell solubilizer was collected, and the LMM and P5CS expressions in the manipulated cell pool were assessed.

[0348] Figure 18 shows Western blot analysis of the generated cell pools, confirming the absence or very low P5CS expression in the host control GS-KO CHO cell line and similar levels of P5CS expression in various LMM pools. This confirms that the P5CS selection system leads to the isolation of cells expressing the P5CS enzyme. Tubulin was used as a loading control. As shown in Figure 18, SCD1 levels were elevated in the medium and high PGK SCD1 CHO pools compared to the low pool, while the progenitor SREBF1 molecule introduced into cells was elevated in the medium and high PGK SREBF1 pools compared to the low pool. Similar observations were made for PGK SCD1 mice and SV40 SREBF1-introduced cells. Therefore, Figure 18 shows that the proline metabolism selection system based on P5CS coupled with LMM expression resulted in the successful isolation of P5CS-expressing cells growing in proline-deficient medium, accompanied by increased LMM levels.

[0349] Subsequently, various LMM expression pools were transfected with recombinant bio-derived therapeutic agents (etanercept and infliximab) and GS vectors, as described in Example 2 above, and then recovered in 96-well plates in proline and glutamine-free medium. Post-transfection cell recovery was observed approximately 14 days after transfection. When more than 75% of the culture was considered confluent (75-80%), the culture in the plate was considered recovered and transferred to a deep-well plate. The cells were then passaged, and the titer was assessed using an Octet instrument. Subsequently, the 10 highest wells from each LMM-transfected pool were combined and grown in a shaking flask. The productivity and growth characteristics were then assessed under fed-batch culture conditions in a miniature bioreactor.

[0350] Figures 19A–19D show plots of Qp against integral viable cell concentration (IVC) for each pool where LMM was mechanically introduced, then the recombinant molecule was transfected, and the productivity and growth in dual cultures on day 9 under fed batch conditions in an ambr® 15 miniature bioreactor were assessed (IVC: time integral of viable cell concentration in culture per hour per milliliter, and Qp: specific productivity as pg / cell / hour).

[0351] The legend at the top of Figure 19A shows (from top to bottom) Control E1, Control E2, SV40-SBF1 High E1, SV40-SBF1 High E2, SV40-SBF1 Medium E1, SV40-SBF1 Medium E2, SV40-SBF1 Low E1, and SV40-SBF1 Low E2. The y-axis starts at 0 from the bottom and progresses to 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6, labeled with Qp (pg / cell / hour). The x-axis starts at 0 from the left and progresses to IVC(10 6The cells are labeled with Qp (pg / cell / hour) and proceed to 500, 1000, 1500, 2000, 2500, 3000 and 3500. The legend at the bottom of Figure 19A shows (from top to bottom) Control I1, Control I2, SV40-SBF1 High I1*, SV40-SBF1 High I2, SV40-SBF1 Medium I1*, SV40-SBF1 Low I1, and SV40-SBF1 Low I2. The y-axis starts from 0 at the bottom and proceeds to 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3, labeled with Qp (pg / cell / hour). The x-axis starts from 0 on the left and proceeds to IVC(10 6 The data is labeled with Qp (pg / cell / hour) and progresses to 500, 1000, 1500, 2000, 2500, 3000, 3500 and 4000. The legend at the top of Figure 19B shows (from top to bottom) Control E1, Control E2, PGK-SBF1 High E1, PGK-SBF1 High E2, PGK-SBF1 Medium E1**, PGK-SBF1 Medium E2, PGK-SBF1 Low E1, and PGK-SBF1 Low E2. The y-axis starts from 0 at the bottom and progresses to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7, labeled with Qp (pg / cell / hour). The x-axis starts from 0 on the left and progresses to IVC(10 6 The cells are labeled with Qp (pg / cell / hour) and proceed to 500, 1000, 1500, 2000, 2500, 3000 and 3500. The legend at the bottom of Figure 19B shows (from top to bottom) Control I1, Control I2, PGK-SBF1 High I2, PGK-SBF1 High I1, PGK-SBF1 Medium I1, PGK-SBF1 Low I2, PGK-SBF1 Low I1, and PGK-SBF1 Medium I2. The y-axis starts from -0.05 from the bottom and proceeds to 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 and 0.35. The x-axis starts from 0 from the left and proceeds to IVC(10 6The data is labeled with Qp (pg / cell / hour) and progresses to 500, 1000, 1500, 2000, 2500, 3000, 3500 and 4000. The legend at the top of Figure 19C shows (from top to bottom) Control E1, Control E2, PGK-MSCD High E1, PGK-mSCD High E2, PGK-mSCD Medium E1, PGK-mSCD Medium E2, PGK-mSCD Low E1, and PGK-mSCD Low E2. The y-axis starts at 0 from the bottom and progresses to 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6, labeled with Qp (pg / cell / hour). The x-axis starts at 0 from the left and progresses to IVC (10 6 The cells are labeled as (x cells / hour / ml) and proceed to 500, 1000, 1500, 2000, 2500, 3000 and 3500. The legend at the bottom of Figure 19C shows (from top to bottom) Control I1, Control I2, PGK-mSCD Medium I2*, PGK-mSCD Low I2, and PGK-mSCD High I1. The y-axis starts from 0 at the bottom and proceeds to 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35 and 0.4, labeled as Qp (pg / cell / hour). The x-axis starts from 0 on the left and proceeds to IVC(10 6 The data is labeled with Qp (pg / cell / hour) and progresses to 500, 1000, 1500, 2000, 2500, 3000, 3500 and 4000. The legend at the top of Figure 19D shows (from top to bottom) Control E1, Control E2, PGK-SCD High E1, PGK-SCD High E2, PGK-SCD Medium E1, PGK-SCD Medium E2, PGK-SCD Low E1, and PGK-SCD Low E2. The y-axis starts from 0 at the bottom and progresses to 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 and 0.45. The x-axis starts from 0 on the left and progresses to IVC(10 6 The data is labeled with Qp (pg / cell / hour) and progresses to 500, 1000, 1500, 2000, 2500, 3000, 3500 and 4000. The legend at the bottom of Figure 19D shows (from top to bottom) Control I1, Control I2, PGK-SCD Medium I1*, PGK-SCD Medium I2, PGK-SCD High I1, PGK-SCD High I2, PGK-SCD Low I1, and PGK-SCD Low I2. The y-axis starts from 0 at the bottom and progresses to 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3, labeled with Qp (pg / cell / hour). The x-axis starts from 0 on the left and progresses to IVC(10 6The cells are labeled at 500, 1000, 1500, 2000, 2500, 3000, 3500, and 4000 (x hours / ml).

[0352] The IVC of control cells (black circles, Figure 19, non-LMM-mediated induction) was generally among the highest IVCs, but in each case, there were several LMM pools that had higher Qp than the control and similar IVCs to the control when expressing models that were difficult to express the protein ("*" indicates that the container was collected on day 12, and "**" indicates that it was collected on day 14). This is consistent with the view that LMM-mediated induction cells produced and cultured using the P5CS selection method can have increased Qp when producing proteins that are difficult to express and IVCs comparable to non-LMM-mediated induction cells under similar conditions.

[0353] Each patent, patent application, and publication cited herein, as well as the disclosures of any patent, patent application, and publication, are incorporated herein by reference in their entirety. Although the present invention is disclosed in relation to a particular aspect, it will be apparent that other aspects and variations of the present invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention. The accompanying claims are intended to be construed as encompassing all such aspects and equivalent variations.

[0354] [Cross-reference of related applications] This application claims priority and interest in U.S. Provisional Patent Application No. 62 / 625773 (filed February 2, 2018), the contents of which said application are incorporated herein by reference in their entirety.

Claims

1. A method for identifying, selecting, or culturing cells containing a target nucleic acid sequence, a) (i) The target nucleic acid sequence and (ii) When expressed, nucleic acid sequences that result in high levels of activity of enzymes in amino acid synthesis pathways, such as the proline synthesis pathway, for example, nucleic acid sequences encoding enzyme molecules containing the said activity and A step of preparing cells containing heterogeneous nucleic acids, such as vectors, such as replicable vectors or integration vectors, b) A step of culturing the cells containing the nucleic acid sequence in the presence of a medium having an insufficient level of the amino acid, such as proline, to support the growth of cells that are the same as the cells that do not have high activity, under conditions sufficient to enable the growth of the cells containing the nucleic acid sequence. A method for identifying, selecting, or culturing cells containing the aforementioned heterogeneous nucleic acid sequences, comprising the above.

2. The method according to claim 1, wherein the heterogeneous nucleic acid includes a vector.

3. The method according to claim 1 or 2, wherein the heterogeneous nucleic acid includes a replicable vector, such as a self-replicating vector.

4. The method according to any one of claims 1 to 3, wherein the heterogeneous nucleic acids include an integration vector.

5. The method according to any one of claims 1 to 4, wherein the culture medium further comprises an inhibitor of the activity of the enzyme.

6. The method according to claim 5, wherein the inhibitor is the amino acid.

7. The method according to claim 5, wherein the inhibitor is not the amino acid.

8. The method according to any one of claims 1 to 7, comprising the step of identifying cells containing heterogeneous nucleic acid sequences.

9. The method according to any one of claims 1 to 8, comprising the step of selecting cells containing heterogeneous nucleic acid sequences.

10. The method according to any one of claims 1 to 9, comprising the step of culturing cells containing heterologous nucleic acid sequences.

11. The method according to any one of claims 1 to 10, wherein the heterogeneous nucleic acid comprises a plurality of vectors (for example, the nucleic acid that yields a high level of activity of the target nucleic acid and enzyme is contained in a plurality of vectors, for example, in different vectors).

12. The method according to any one of claims 1 to 11, wherein the heterogeneous nucleic acids include a plurality of integration vectors (for example, the nucleic acids that result in a high level of activity of the target nucleic acid and enzyme are included in the plurality of integration vectors, for example, in different vectors).

13. The method according to any one of claims 1 to 12, wherein the heterogeneous nucleic acid comprises a plurality of self-replicating vectors (for example, the nucleic acid that yields a high level of activity of the target nucleic acid and enzyme is contained in the plurality of self-replicating vectors, for example, in different vectors).

14. The method according to any one of claims 1 to 13, wherein the heterogeneous nucleic acids are integrated into the genome of the cell, for example, the chromosomal genome.

15. The method according to any one of claims 1 to 14, wherein the amino acid includes a natural amino acid.

16. The method according to any one of claims 1 to 15, wherein the amino acid includes the amino acids listed in Table 1.

17. The method according to claim 16, wherein the amino acid is alanine.

18. The method according to claim 16, wherein the amino acid is leucine.

19. The method according to claim 16, wherein the amino acid is isoleucine.

20. The method according to claim 16, wherein the amino acid is methionine.

21. The method according to claim 16, wherein the amino acid is valine.

22. The method according to claim 16, wherein the amino acid is phenylalanine.

23. The method according to claim 16, wherein the amino acid is asparagine.

24. The method according to claim 16, wherein the amino acid is cysteine.

25. The method according to claim 16, wherein the amino acid is glutamine.

26. The method according to claim 16, wherein the amino acid is serine.

27. The method according to claim 16, wherein the amino acid is threonine.

28. The method according to claim 16, wherein the amino acid is aspartic acid.

29. The method according to claim 16, wherein the amino acid is glutamic acid.

30. The method according to claim 16, wherein the amino acid is arginine.

31. The method according to claim 16, wherein the amino acid is histidine.

32. The method according to claim 16, wherein the amino acid is lysine.

33. The method according to claim 16, wherein the amino acid is glycine.

34. The method according to any one of claims 1 to 16, wherein the amino acid is selected from proline and tyrosine and tryptophan.

35. The method according to claim 34, wherein the amino acid is proline.

36. The method according to claim 34, wherein the amino acid is tyrosine.

37. The method according to claim 34, wherein the amino acid is tryptophan.

38. The method according to any one of claims 5 to 37, wherein the inhibitor binds to the enzyme, for example, the inhibitor binds to the enzyme and inhibits it.

39. The method according to any one of claims 5 to 38, wherein the inhibitor inhibits the transcription of the enzyme.

40. The method according to any one of claims 5 to 39, wherein the inhibitor inhibits the translation of the enzyme.

41. The method according to any one of claims 5 to 40, wherein the inhibitor comprises a nucleic acid, such as RNA, such as antisense or siRNA.

42. The method according to any one of claims 5 to 41, wherein the inhibitor comprises an aptamer.

43. The method according to any one of claims 5 to 42, wherein the inhibitor comprises a small molecule.

44. The method according to any one of claims 5 to 43, wherein the inhibitor is an analog of the substrate of the enzyme.

45. The method according to any one of claims 5 to 44, wherein the inhibitor is an analog of the amino acid, for example, an analog of proline, tyrosine, or tryptophan.

46. The method according to any one of claims 5 to 45, wherein the inhibitor includes a competitive inhibitor.

47. The method according to any one of claims 5 to 46, wherein the inhibitor inhibits the rate-limiting enzyme for the synthesis of the amino acid, for example, the inhibitor inhibits the rate-limiting enzyme for the synthesis of the amino acid in the culture medium.

48. The method according to any one of claims 1 to 47, wherein the amino acid comprises proline.

49. The method according to any one of claims 1 to 48, wherein the enzyme comprises a pyrroline-5-carboxylic acid synthase (P5CS) molecule.

50. The method according to any one of claims 1 to 49, wherein the heterogeneous nucleic acid sequence includes a sequence encoding a P5CS molecule.

51. The method according to any one of claims 5 to 50, wherein the inhibitor is a substrate of the enzyme, or an analog, variant, or derivative thereof.

52. The method according to any one of claims 1 to 51, wherein the inhibitor is an analog of proline, for example, L-azetidine-2-carboxylic acid, 3,4-dehydro-L-proline, or L-4-thiazolidinedcarboxylic acid.

53. The method according to any one of claims 5 to 52, wherein the amino acid comprises proline, and the enzyme and inhibitor are selected from Table 1.

54. The method according to any one of claims 5 to 16, 34, and 38 to 47, wherein the amino acid comprises tyrosine, and the enzyme and / or inhibitor is selected from Table 1.

55. The method according to any one of claims 5 to 16, 34, 38 to 47, and 54, wherein the heterogeneous nucleic acid sequence includes a sequence encoding a polypeptide and / or an inhibitor of a tyrosine biosynthesis enzyme.

56. The method according to claim 54 or 55, wherein the inhibitor is an analog of the substrate of the enzyme.

57. The method according to any one of claims 5 to 16, 34, 38 to 47, and 54 to 56, wherein the inhibitor is a tyrosine analog.

58. The method according to any one of claims 5 to 16, 34, 38 to 47, and 54 to 57, wherein the amino acid comprises tyrosine, and the enzyme and inhibitor are selected from Table 1.

59. The method according to any one of claims 5 to 16, 34, and 38 to 47, wherein the amino acid comprises tryptophan, and the enzyme and / or inhibitor is selected from Table 1.

60. The method according to any one of claims 5 to 16, 34, 38 to 47, and 59, wherein the heterogeneous nucleic acid sequence includes a sequence encoding a polypeptide and / or an inhibitor of tryptophan biosynthesis enzyme.

61. The method according to claim 59 or 60, wherein the inhibitor is an analog of the substrate of the enzyme.

62. The method according to any one of claims 5 to 16, 34, 38 to 47 and 59 to 61, wherein the inhibitor is an analog of tryptophan.

63. The method according to any one of claims 5 to 16, 34, 38 to 47, and 59 to 62, wherein the amino acid comprises tryptophan, and the enzyme and inhibitor are selected from Table 1.

64. The method according to any one of claims 1 to 63, comprising the step of culturing cells that do not have high enzyme activity together with the cells that have high activity.

65. The method according to any one of claims 1 to 64, wherein the highly active cells grow more rapidly than the non-active cells, for example, by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 times faster.

66. The method according to any one of claims 1 to 65, wherein less than about 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 percent of the selected, for example, cells selected based on growth, lack the nucleic acid.

67. The method according to any one of claims 1 to 66, further comprising the step of selecting cells that show growth.

68. The method according to any one of claims 1 to 67, wherein the cells include eukaryotic cells.

69. The method according to any one of claims 1 to 68, wherein the cells include animal cells.

70. The method according to any one of claims 1 to 69, wherein the cells include mammalian cells.

71. The method according to any one of claims 1 to 70, wherein the cells include rodent cells.

72. The method according to any one of claims 1 to 71, wherein the cells include CHO cells.

73. The method according to any one of claims 1 to 72, wherein the cells include GSKO CHO cells.

74. The method according to any one of claims 1 to 73, wherein the endogenous copy of the sequence encoding the enzyme is inactivated, for example, by the deletion of a structural or regulatory region.

75. The method according to any one of claims 1 to 74, wherein an endogenous copy of the sequence encoding a second amino acid synthase is inactivated, for example, by deletion of a structural or regulatory region.

76. The method according to any one of claims 1 to 75, wherein an endogenous copy of the sequence encoding GS is inactivated, for example, by deletion of a structural or regulatory region.

77. The method according to any one of claims 1 to 76, wherein the target nucleic acid sequence encodes a peptide molecule.

78. The method according to any one of claims 1 to 77, wherein the target nucleic acid sequence is heterogeneous.

79. The method according to any one of claims 1 to 78, wherein a different peptide molecule is selected from any of Tables 5 to 8.

80. The method according to any one of claims 1 to 79, comprising the step of selecting cells that grow in the culture medium.

81. The method according to claim 80, wherein the culture medium contains an inhibitor.

82. 1) A step of selecting cells that grow in the culture medium, 2) The step of culturing the cells in a second medium, for example, under a second set of culture conditions, or subjecting the selected cells to a second selection. The method according to any one of claims 1 to 81, including the method described in that claim.

83. 1) The culture medium contains the inhibitor, 2) The second culture medium contains the inhibitor, The method according to claim 82.

84. 1) A step of selecting cells that grow in the culture medium, 2) The step of culturing the cells in a second medium, for example, under a second set of culture conditions, or subjecting the selected cells to a second selection. The method according to any one of claims 1 to 83, wherein the concentration of the inhibitor in one of the culture media (1) and 2) is greater than the concentration of the inhibitor in the other of the culture media (1) and 2).

85. The method according to claim 84, wherein the concentration of the inhibitor in 1) is greater than the concentration of the inhibitor in 2).

86. The method according to claim 84, wherein the concentration of the inhibitor in (2) is greater than the concentration of the inhibitor in (1).

87. The method according to any one of claims 84 to 86, wherein the culture medium in the step having a lower concentration of the inhibitor is essentially free of the inhibitor.

88. The method according to any one of claims 1 to 87, wherein the nucleic acid sequence of (ii) is operably linked to a control sequence, for example, a promoter.

89. The method according to claim 88, wherein the control sequence comprises a sequence selected from SV40, mCMV, hCMV, or PGK promoter or variants thereof.

90. The method according to claim 88, wherein the control sequence controls the expression of LMM.

91. The method according to claim 90, wherein the control array and LMM are as listed in any single row of Table 3.

92. The method according to claim 90, wherein the control sequence includes an SV40 promoter and the LMM is mouse SCD1.

93. The method according to claim 90, wherein the control sequence includes an SV40 promoter, and the LMM is SCD1, for example, CHO SCD1.

94. The method according to claim 90, wherein the control sequence includes an SV40 promoter, and the LMM is SREBF1, for example, CHO SREBF1.

95. The method according to claim 90, wherein the control sequence includes an SV40 promoter, and the LMM is SREB411, for example, CHO SREB411.

96. The method according to claim 90, wherein the control sequence includes an mCMV promoter and the LMM is mouse SCD1.

97. The method according to claim 90, wherein the control sequence includes an mCMV promoter, and the LMM is SCD1, for example, CHO SCD1.

98. The method according to claim 90, wherein the control sequence includes an mCMV promoter, and the LMM is SREBF1, for example, CHO SREBF1.

99. The method according to claim 90, wherein the control sequence includes an mCMV promoter, and the LMM is SREB411, for example, CHO SREB411.

100. The method according to claim 90, wherein the control sequence includes an hCMV promoter and the LMM is mouse SCD1.

101. The method according to claim 90, wherein the control sequence includes an hCMV promoter, and the LMM is SCD1, for example, CHO SCD1.

102. The method according to claim 90, wherein the control sequence includes an hCMV promoter, and the LMM is SREBF1, for example, CHO SREBF1.

103. The method according to claim 90, wherein the control sequence includes an hCMV promoter, and the LMM is SREB411, for example, CHO SREB411.

104. The method according to claim 90, wherein the control sequence includes a PGK promoter and the LMM is mouse SCD1.

105. The method according to claim 90, wherein the control sequence includes a PGK promoter and the LMM is SCD1, for example, CHO SCD1.

106. The method according to claim 90, wherein the control sequence includes a PGK promoter, and the LMM is SREBF1, for example, CHO SREBF1.

107. The method according to claim 90, wherein the control sequence includes a PGK promoter, and the LMM is SREB411, for example, CHO SREB411.

108. The method according to claim 88, wherein the control sequence controls the expression of LMM, and the culture medium contains an inhibitor of the activity of the enzyme.

109. The method according to claim 108, wherein the control sequence, LMM and inhibitor (e.g., a P5CS inhibitor) are as listed in any single row of Table 4.

110. The method according to any one of claims 88 to 109, wherein the culture medium contains L-azetidine-2-carboxylic acid.

111. The method according to any one of claims 88 to 110, wherein the culture medium comprises 3,4-dehydro-L-proline.

112. The method according to any one of claims 88 to 111, wherein the culture medium contains L-4-thiazolidinedic acid.

113. The method according to any one of claims 1 to 112, further comprising the step of recovering the product of the target nucleic acid sequence.

114. The method according to any one of claims 1 to 113, further comprising the step of recovering the product from the culture medium.

115. The method according to any one of claims 1 to 114, further comprising the step of recovering the product from the cells.

116. i) A step of selecting cells to grow in the culture medium, ii) The step of culturing the cells in a second medium, for example, under a second set of culture conditions, for example, subjecting the selected cells to a second selection, iii) A step of recovering the product from the cells or the second culture medium. The method according to any one of claims 1 to 115, including the method described in that claim.

117. A method for identifying, selecting, or culturing cells containing a target nucleic acid sequence, a) (i) The target nucleic acid sequence and (ii) When expressed, nucleic acid sequences that result in high levels of activity of enzymes in amino acid synthesis pathways, such as the proline synthesis pathway, for example, nucleic acid sequences encoding enzyme molecules containing the said activity and The steps include: preparing cells containing nucleic acids, such as vectors, such as replicable vectors; b) A step of culturing the cells containing the nucleic acid sequence in a first medium (and optionally containing an enzyme inhibitor) having a level of the amino acid, such as proline, that is insufficient to support the growth of cells that are the same as the target cells that do not have high activity, under conditions sufficient to enable the growth of the cells containing the nucleic acid sequence, c) A step of culturing the cells containing the nucleic acid sequence in the presence of a second medium (and optionally the second medium containing an inhibitor of the second enzyme) under conditions sufficient to enable the growth of the cells containing the nucleic acid sequence, in the presence of a second amino acid, such as tyrosine, at a level insufficient to support the growth of cells that are the same as the target cells that do not have high activity. A method for identifying, selecting, or culturing cells containing heterogeneous nucleic acid sequences, including the above.

118. The method according to claim 117, wherein step b is initiated before step c begins.

119. The method according to claim 117 or 118, wherein step b is performed before step c.

120. The method according to claim 117 or 118, wherein steps b and c are performed simultaneously.

121. The method according to any one of claims 117 to 120, wherein steps b and c are performed in the same culture medium.

122. The method according to any one of claims 117 to 121, wherein steps b and c are performed in the same container.

123. The selection in step b and the selection in step c are performed in the same culture medium, and the culture medium is (a) Having an insufficient level of the amino acid, for example proline, to support the growth of cells that are the same as the target cells that do not have high activity, (b) Having the same level of the second amino acid, e.g., tyrosine, as the target cells which do not have high activity of the second enzyme, The method according to any one of claims 117 to 122.

124. The aforementioned cells, (iii) A nucleic acid sequence that, when expressed, results in a high level of activity of the second enzyme in the amino acid synthesis pathway, for example, a nucleic acid sequence encoding an enzyme molecule containing the said activity. The method according to any one of claims 117 to 123, further comprising:

125. The method according to any one of claims 117 to 124, wherein the cells are cultured in a medium having an inhibitor of the activity of the enzyme.

126. The method according to any one of claims 117 to 125, wherein the cells are cultured in a medium having an inhibitor of the activity of the second enzyme.

127. The method according to any one of claims 117 to 126, wherein the second enzyme is located in the same pathway as the enzyme in (ii), for example, in the proline synthesis pathway.

128. The second enzyme is present in the same amino acid synthesis pathway and is, for example, a nucleic acid sequence encoding an enzyme molecule containing the aforementioned activity. b) The culture medium is iv) Inhibitors of the activity of the second enzyme Further including, The method according to claim 127.

129. The method according to any one of claims 117 to 128, wherein the second enzyme is located in a different pathway from the enzyme in (ii), for example, the enzyme is located in the proline synthesis pathway and the second enzyme is located in a pathway other than proline, for example, the tyrosine or tryptophan pathway.

130. The second enzyme is, for example, a nucleic acid sequence encoding an enzyme molecule containing the activity present in the synthesis pathway of the second amino acid. b) The culture medium is iii) The second amino acid, for example, an amino acid other than proline, at a level insufficient to support the growth of cells that are the same as the target cells that do not have high activity, iv) Inhibitors of the activity of the second enzyme and Further including, The method according to claim 129.

131. Cells containing a sequence that includes a regulatory region from any of the following: an SV40 promoter sequence, an mCMV promoter sequence, or a PGK promoter sequence, for example, a sequence that encodes a heterologous lipid metabolism modifier (LMM) operably linked to a promoter sequence.

132. The cell according to claim 131, wherein the LMM modifies the pathways listed in Table 2.

133. The cell according to claim 131 or 132, wherein the LMM comprises sterol-CoA desaturase-1 (SCD1).

134. The cell according to claim 131 or 132, wherein the LMM comprises sterol regulatory element-binding transcription factor 1 (SREBF1).

135. The cell according to claim 131 or 132, wherein the LMM comprises a cleaved isoform of SREBF1 (for example, a cleaved isoform of SREBF1 lacking a regulatory domain, e.g., SREB411).

136. The cell according to any one of claims 131 to 135, wherein the sequence encoding a heterologous lipid metabolism modifier (LMM) is arranged in the vector.

137. The cell according to any one of claims 131 to 136, wherein the vector further comprises a sequence encoding a selectable marker, for example, a marker that, if present, enables survival or growth in a specified culture medium.

138. The cell according to any one of claims 131 to 137, wherein the vector further comprises a sequence encoding a selectable marker that, if present, enables survival or growth in a medium lacking nutrients, such as amino acids.

139. The cell according to claim 138, wherein the nutrient is an amino acid, for example, an amino acid selected from the amino acids listed in Table 1.

140. The cell according to claim 139, wherein the nutrient comprises proline.

141. The cell according to any one of claims 137 to 140, wherein the marker comprises a pyrroline-5-carboxylic acid synthase (P5CS) molecule.

142. The cell according to any one of claims 138 to 141, wherein the nutrient comprises tyrosine.

143. The cell according to any one of claims 138 to 142, wherein the nutrient comprises tryptophan.

144. The cell according to any one of claims 131 to 143, wherein the marker is selected from Table 1.

145. The cell according to any one of claims 137 to 144, wherein the marker comprises glutamine synthetase.

146. The cell according to any one of claims 131 to 145, wherein the sequence encoding the LMM is operably linked to the sequence encoding the SV40 promoter sequence.

147. The cell according to any one of claims 131 to 145, wherein the sequence encoding the LMM is operably linked to a sequence encoding the mCMV promoter sequence.

148. The cell according to any one of claims 131 to 145, wherein the sequence encoding the LMM is operably linked to the sequence encoding the PGK promoter sequence.

149. The cell according to any one of claims 131 to 148, wherein the LMM is SCD1.

150. The cell according to claim 149, wherein the LMM is mouse SCD1.

151. The cell according to claim 149, wherein the LMM is CHO SCD1.

152. The cell according to any one of claims 131 to 148, wherein the LMM is SREBF1.

153. The cell according to any one of claims 131 to 148, wherein the LMM is CHO SREBF1.

154. The cell according to any one of claims 131 to 148, wherein the LMM is SREB411.

155. The cell according to any one of claims 131 to 148, wherein the LMM is CHO SREB411.

156. The cell according to any one of claims 131 to 148, wherein the LMM comprises sterol-CoA desaturase-1 (SCD1) and the promoter comprises an SV40 promoter sequence.

157. The cell according to any one of claims 131 to 148, wherein the LMM comprises sterol-CoA desaturase-1 (SCD1), and the promoter comprises an mCMV promoter sequence.

158. The cell according to any one of claims 131 to 148, wherein the LMM comprises sterol-CoA desaturase-1 (SCD1) and the promoter comprises a PGK promoter sequence.

159. The cell according to any one of claims 131 to 148, wherein the LMM comprises sterol regulatory element-binding transcription factor 1 (SREBF1), and the promoter comprises an SV40 promoter sequence.

160. The cell according to any one of claims 131 to 148, wherein the LMM comprises sterol regulatory element-binding transcription factor 1 (SREBF1), and the promoter comprises an mCMV promoter sequence.

161. The cell according to any one of claims 131 to 148, wherein the LMM comprises sterol regulatory element-binding transcription factor 1 (SREBF1), and the promoter comprises a PGK promoter sequence.

162. The cell according to any one of claims 131 to 148, wherein the LMM comprises SREB411 and the promoter comprises an SV40 promoter sequence.

163. The cell according to any one of claims 131 to 148, wherein the LMM comprises SREB411 and the promoter comprises an mCMV promoter sequence.

164. The cell according to any one of claims 131 to 148, wherein the LMM comprises the sterol SREB411 and the promoter comprises a PGK promoter sequence.

165. The cells according to any one of claims 131 to 148, wherein the culture medium comprises an inhibitor listed in Table 1 or 4.

166. The cells according to any one of claims 131 to 148, wherein the culture medium contains a P5CS inhibitor.

167. The cell according to claim 166, wherein the P5CS inhibitor is L-azetidine-2-carboxylic acid.

168. The cell according to claim 166, wherein the P5CS inhibitor is 3,4-dehydro-L-proline.

169. The cell according to claim 166, wherein the P5CS inhibitor is L-4-thiazolidinediocarboxylic acid.

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