Improved cells and cell cultures

Abstract

Cells having improved growth and related cell cultures are provided. In some aspects, cells provided herein have increased expression of thioredoxin interacting protein (TXNIP).

Description

IMPROVED CELLS AND CELL CULTURESREFERENCE TO SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on April 3, 2024, is named PC072975_SeqListing_ST.26.xml and is 4 kilobytes in size.BACKGROUND

[0002] Large molecule biologies (e.g. recombinant proteins) are typically produced in cultured host cells, such has Chinese Hamster Ovary Cells (CHO) cells or human embryonic kidney (HEK) cells. While major advances have been made in recent decades in the development of improved host cells for biologies production, the generation and selection of specific host cells that have desirable growth and productivity characteristics has remained a time-consuming and challenging process.

[0003] Accordingly, there is a need for host cells having robust growth characteristics, and a need for methods of generating and selecting such cells.SUMMARY

[0004] In some aspects, the disclosure provides herein a mammalian host cell comprising an exogenous thioredoxin interacting protein (TXNIP) gene. In some embodiments, a cell is a mouse cell, a rat cell, a Chinese Hamster Ovary (CHO) cell or a human cell. In some embodiments, a cell further comprises an exogenous gene encoding a therapeutic molecule or portion thereof. In some embodiments, provided herein is a CHO host cell comprising an exogenous thioredoxin interacting protein (TXNIP) gene and an exogenous gene encoding a therapeutic molecule or portion thereof. In some embodiments, a therapeutic molecule is a recombinant protein. In some embodiments, a recombinant protein is selected from the group consisting of an antibody or fragment thereof, nanobody, glycoprotein, growth factor, clotting factor, cytokine, fusion protein, pharmaceutical drug substance, vaccine, enzyme, receptor, hormone, regulatory factor, antigen, binding agent and detectable protein. In some embodiments, a recombinant protein is an antibody. In some embodiments, a TXNIP gene is chromosomally-integrated in a host cell chromosome, optionally a CHO cell chromosome. In some embodiments, a host cell chromosome contains a recombination target site for sitespecific integration of the exogenous TXNIP gene in a host cell chromosome. In some embodiments, a nucleotide sequence encoding an exogenous TXNIP gene encodes a polypeptide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 , comprises a polynucleotide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 2, or both.

[0005] In some aspects, the disclosure provides herein a mammalian host cell comprising an exogenous thioredoxin interacting protein (TXNIP) gene and an exogenous gene encoding a therapeutic molecule or portion thereof, wherein the cell has one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic compared to an otherwise identical cell that does not contain an exogenous TXNIP gene, and optionally wherein the host cell is a CHO cell. In some embodiments, an improved growth characteristic is selected from cell count, cell viability, viable cell count, cell density, viable cell density, cell metabolism, cell size or a combination thereof. In some embodiments, cell metabolism is a metabolic shift by the cell, and optionally wherein the metabolic shift comprises an increased lactate consumption, a decreased lactate production or a combination thereof. In some embodiments, a nucleotide sequence encoding an exogenous TXNIP gene encodes a polypeptide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 , comprises a polynucleotide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 2, or both.

[0006] In some aspects, the disclosure provides herein a host cell culture comprising a host cell as disclosed herein, wherein the host cell is a CHO cell, and wherein the host cell culture has one or more of 1) an increased viable cell density and 2) exhibits a metabolic shift, as compared to a viable cell density or metabolic shift of a host cell culture comprising an otherwise identical cell that does not comprise an exogenous TXNIP gene. In some embodiments, a metabolic shift comprises an increased lactate consumption, a decreased lactate production or a combination thereof. In some embodiments, an increased lactate consumption, a decreased lactate production or combination thereof is detected by measuring lactate concentration in media of a cell culture. In some embodiments, a lactate concentration in media of a cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least90 fold, at least 95 fold at least 100 fold lower than a lactate concentration in media of a second cell culture comprising an otherwise identical host cell that does not comprise an exogenous TXNIP gene. In some embodiments, a viable cell density of a cell culture is increased by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to a viable cell density of a second cell culture comprising an otherwiseidentical host cell that does not comprise an exogenous TXNIP gene.

[0007] In some aspects, provided herein are methods of selecting a mammalian host cell having one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic, the method comprising: a) assaying a mammalian host cell for at least one of 1 ) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity; b) selecting the host cell comprising at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity, wherein the host cell comprising at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity has one or more of reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic as compared to synthesis of a growth inhibitor, synthesis of a productivity inhibitor or a growth characteristic of an otherwise identical cell that does not comprise at least one of 1 ) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity. In some embodiment an improved growth characteristic is an increased viable cell density when a host cell is grown in a cell culture. In some embodiments, a growth inhibitor or productivity inhibitor is a cell metabolite, optionally lactate.

[0008] In some aspects, provided herein are methods of preparing a mammalian host cell having one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic, the method comprising: introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP into the host cell, wherein the host cell comprising the exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP has one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic compared to an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP. In some embodiments, a method further comprises introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a therapeutic molecule into the host cell and culturing the host cell to produce a first host cell culture. In some embodiments, an amount of therapeutic molecule produced by a first host cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than an amount of therapeutic molecule produced by a second host cell culture comprising an otherwise identical mammalian host cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP. In some embodiments, a therapeutic molecule is a recombinantprotein and at least 0.01 grams protein per liter cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1 .2 g / L, 1 .4 g / L, 1 .6 g / L, 1 .8 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L. 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L or 20 g / L is produced by a first host cell culture. In some embodiments, a recombinant protein is an antibody, and optionally a bispecific antibody.

[0009] In some embodiments, a first host cell culture has one or more of: 1 ) an increased viable cell density and 2) exhibits a metabolic shift as compared to a viable cell density or metabolic shift of a second host cell culture comprising an otherwise identical mammalian host cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP. In some embodiments, a metabolic shift comprises an increased lactate consumption, a decreased lactate production or a combination thereof. In some embodiments, an increased lactate consumption, decreased lactate production or combination thereof is detected by measuring lactate concentration in media of a first host cell culture. In some embodiments, a lactate concentration in media of a first host cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold lower than a lactate concentration in media of a second host cell culture comprising an otherwise identical mammalian host cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP. In some embodiments, a lactate concentration in media of a first host cell culture is less than 0.01 grams lactate per liter cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 1 1 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L. In some embodiments, a viable cell density of a first host cell culture is increased by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to a viable cell density of a second host cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP. In some embodiments, a viable cell density of a first host cell culture is at least 5 x 105cells per milliliter cell culture media (cells / mL), 10 x 105cells / mL, 25 x 105cells / mL, 50 x 105cells / mL, 100 x 105cells / mL, 150 x 105cells / mL, 200 x 105cells / mL, 250 x 105cells / mL, 300 x 105cells / mL, 350 x 105cells / mL, 400 x 105cells / mL, 450 x 5005cells / mL, 550 x 105cells / mL, 600 x 105cells / mL, 650 x 105cells / mL, 700 x 105cells / mL, 750 x 105cells / mL, 800 x 105cells / mL, 850 x 105cells / mL, 900 x 105cells / mL, 950 x 105cells / mL or 1000 x 105cells / mL. In some embodiments, a nucleotide sequence encoding an exogenous TXNIP gene encodes a polypeptide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 1 , comprises a polynucleotide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 2, or both.

[0010] In some aspects, as provided herein, a host cell as disclosed herein, a host cell culture as disclosed herein, or both are contacted with a molecule that increases expression of a TXNIP gene or TXNIP polypeptide. In some embodiments, a molecule that increases expression of a TXNIP gene or a TXNIP polypeptide is glucosamine, a molecule that inhibits expression of the MYC gene, a molecule that inhibits MYC activity or a combination thereof.

[0011] In some aspects, as provided herein are methods for producing a recombinant therapeutic molecule or portion thereof, the method comprising: a) providing a host cell as disclosed herein, a host cell culture as disclosed herein or a host cell selected or prepared by a method as disclosed herein and b) culturing the host cell or host cell culture under conditions sufficient to produce the recombinant therapeutic molecule. In some embodiments, a method for producing a recombinant therapeutic molecule or portion thereof further comprises recovering the recombinant therapeutic molecule from the cell or cell culture.

[0012] In some aspects, as provided herein are methods for producing a metabolic shift in a host cell, the method comprising 1) introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP into the host cell, 2) contacting a host cell comprising a TXNIP gene with a molecule that increases expression of a TXNIP gene in the cell, increases expression of a TXNIP polypeptide in the cell, increases TXNIP activity in the cell or a combination thereof, or 3) both, and optionally, wherein the host cell is a CHO cell. In some embodiments, a metabolic shift comprises an increased lactate consumption, a decreased lactate production or a combination thereof. In some embodiments, a method for producing a metabolic shift in a host cell, further comprises culturing the host cell in a cell culture, and measuring lactate concentration in media of the host cell culture. In some embodiments, a lactate concentration in media of a host cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold lower than a lactate concentration in media of a second host cell culture comprising an otherwise identical host cell that 1 ) does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP, 2) is not contacted with a molecule that increases expression of a TXNIP gene in the cell, increases expression of a TXNIP polypeptide in the cell, increases TXNIP activity in the cell or a combination thereof or 3) both. In some embodiments, a lactate concentration in media of a host cell culture exhibiting a metabolic shift is less than 0.01 grams lactate per liter cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L. in some embodiments, a molecule that increases expression of the TXNIP gene in a cell, increases expression of the TXNIP polypeptide in a cell, increases TXNIP activity in a cell or a combination thereof, is selected from the group consisting of glucosamine, an inhibitor of MYC gene expression, an inhibitor of MYC activity and a combination thereof.

[0013] In some embodiments, provided herein is a cell, cell culture, composition or method as exemplified by any of the Examples provided herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1A-1C: depict exemplary growth kinetics, metabolic characteristics and TXNIP gene expression of cell line A and B in fedbatch cultures. Duplicate fedbatch cultures were performed for each cell line. (FIG. 1A) Viable cell density, (FIG. 1 B) Lactate and (FIG. 1 C) TXNIP gene expression. Black filled circles - cell line A and grey filled squares - cell line B.

[0015] FIG. 2A-2D: depict exemplary growth kinetics, metabolic characteristics and TXNIP gene and protein expression of cell line C and D in fedbatch cultures. (FIG. 2A) Viable cell density, (FIG. 2B) Lactate (FIG. 2C) TXNIP gene expression and (FIG. 2D) Western blot for TXNIP and B-ACTIN (housekeeping control). Black filled circles - cell line C and grey filled squares - cell line D.

[0016] FIG. 3: depict exemplary MYC gene expression of cell line A and B in fedbatch cultures. Black filled circles - cell line A and grey filled squares - cell line B.

[0017] FIG. 4A-4D: depict an exemplary effect of MYC inhibitor treatment on growth kinetics, metabolic characteristics, TXNIP and MYC gene expression of cell line B in ambr15 fedbatch cultures. Cell line B was treated with 0.4 micromolar of MYC inhibitor JQ1 , 2.5 micromolar of MYC inhibitor OTX-015 or DMSO (control) on days 4 and 6. Duplicate fedbatch cultures were performed for condition. (FIG. 4A) Viable cell density. (FIG. 4B) Lactate. (FIG. 4C) Fold change in MYC mRNA. Fold change was calculated with respect to MYC expression in control condition on day 0. (FIG. 4D) Fold change in TXNIP mRNA. Fold change was calculated with respect to TXNIP expression in control condition on day 0. Black filled circles - JQ1 treatment condition, black filled triangles - OTX-15 treatment condition, and grey filled squares - DMSO treatment (control) condition.

[0018] FIG. 5A-5H: depict an exemplary effect of MYC inhibitor treatment on growth kinetics, metabolic characteristics, TXNIP and MYC gene expression of cell lines A and B fedbatch cultures executed in 1 L working volume glass bioreactors. Cell line A was treated with 0.25 micromolar MYC inhibitor JQ1 or DMSO (control) on days 4 and 5. (FIG. 5A) Viable cell density. (FIG. 5B) Lactate. (FIG. 5C) Fold change in MYC mRNA. Fold change calculated withrespect to MYC expression in control condition on day 0. (FIG. 5D) Fold change in TXNIP mRNA. Fold change calculated with respect to TXNIP expression in control condition on day 0. Black filled circles - JQ1 treatment condition and grey filled squares - DMSO treatment (control) condition. Cell line B was treated with 0.25 micromolar JQ1 or DMSO (control) on days 4, 5, 7, 8 and 9. (FIG. 5E) Viable cell density. (FIG. 5F) Lactate. (FIG. 5G) Fold change in MYC mRNA. Fold change calculated with respect to MYC expression in control condition on day 0. (FIG. 5H) Fold change in TXNIP mRNA. Fold change calculated with respect to TXNIP expression in control condition on day 0. Black filled circles - JQ1 treatment condition and grey filled squares - DMSO treatment (control) condition.

[0019] FIG. 6A-6C: depict an exemplary time course of intracellular levels of glucosamine related intermediates in fedbatch cultures of cell line A and B. (A) N-acetylglucosamine 6- phosphate intracellular levels. (B) N-acetylglucosamine / N-acetylgalactosamine. (C) N-acetylglucosamine 1 -phosphate. Black filled circles - cell line A and grey filled squares - cell line B.

[0020] FIG. 7A-7I: depict an exemplary effect of treatment with glucosamine, JQ1 or combination of both on growth and metabolic characteristics of CHO cell line B, E and F. All three cell lines were treated with glucosamine, JQ1 or glucosamine + JQ1 . Cell line B and E were treated with glucosamine (10 mM) and JQ1 (0.4 micromolar) treatment were performed on days 4, 6 and 8. For cell line F, glucosamine treatment were done days 4 (10 mM), 7 (10 mM), 9 (5 mM) and 10 (5 mM) whereas JQ1 treatment was done on day 4 (0.4 micromolar). Cell line B viable cell density (FIG. 7A), lactate (FIG. 7B) and TXNIP fold change (w.r.t control day 3) (FIG. 7C). Cell line E viable cell density (FIG. 7D), lactate (FIG. 7E) and TXNIP fold change (w.r.t control day 3) (FIG. 7F). Cell line F viable cell density (FIG. 7G), lactate (FIG. 7H) and TXNIP fold change (w.r.t control day 3) (FIG. 7I). Black filled squares - DMSO treatment (control) condition, black filled circles - glucosamine treatment condition, black filled diamonds - JQ1 treatment condition and black filled triangles - glucosamine and JQ1 treatment condition.

[0021] FIG. 8: depicts an exemplary schematic representation of transfection of TXNIP in CHO cell line G. Constitutive vectors harboring either the LACZ (control) gene or TXNIP gene were transfected into cell line G and the pools were recovered for growth in G418 selection pressure.

[0022] FIG. 9A-9D: depict exemplary growth and viability recovery profiles of TXNIP and LACZ pools and expression levels of mouse TXNIP and endogenous (CHO) TXNIP in the recovered pools. (FIG. 9A) Viable cell density. (FIG. 9B) Percent viability. (FIG. 9C) Mouse TXNIP relative expression to B-ACTIN. (FIG. D) Endogenous TXNIP relative expression to B- ACTIN. Grey filled squares - Topo LACZ (control) gene, black filled circles - Topo TXNIP, grey filled diamonds - mock transfected.

[0023] FIG. 10A-10C: depict exemplary growth kinetics and metabolic characteristics ofrecovered TXNIP and LACZ pools. (FIG. 10A) Viable cell densities. Average VCDs of four TXNIP and LACZ pools plotted. (FIG. 10B) Lactate. Average lactate profiles of four TXNIP and LACZ pools plotted. (FIG. 10C) Glucose. Average glucose profiles of four TXNIP and LACZ pools plotted. Black filled circles - TXNIP pool average and grey filled squares - LACZ pool average.

[0024] FIG. 11 : depicts exemplary mouse TXNIP expression levels in single cell clones obtained using limited dilution method. TXNIP expression levels plotted using DCT against B- ACTIN.

[0025] FIG. 12: depicts exemplary specific lactate production rate of single cell clones with or without mouse TXNIP expression and cell line G (wildtype, WT) across multiple passages in maintenance cultures. Black filled circles - clones expressing mouse TXNIP and grey filled squares - clones with no mouse TXNIP expression or cell line G (WT).

[0026] FIG. 13A-13D: depict exemplary growth kinetics and metabolic characteristics of cell line G (WT), WT with treatment (with OTX-015 and glucosamine), and clones with high or no expression of mouse TXNIP. (FIG. 13A) Viable cell density. (FIG. 13B) Lactate. (FIG. 13C) Glucose. (FIG. 13D) Moving average specific lactate production rate. Black filled circles - clones with higher overexpression of mouse TXNIP gene, grey filled squares - WT and clones with no mouse TXNIP expression, and light grey filled diamonds - WT treated with OTX-015 (MYC inhibitor) and glucosamine.

[0027] FIG. 14A-14D: depict exemplary growth kinetics and metabolic characteristics of a WT cell line and P4P3F4 clone transfected with and expressing mouse TXNIP gene grown under three conditions: 4-6 g / L glucose (black circles), 1-2 g / L glucose (black diamonds) and HiPDOG conditions (black squares). FIG. 14A, viable cell density (cells / mL); FIG. 14B, lactate (g / L); FIG. 14C, glucose (g / L); FIG. 14D, IgG production (g / L).

[0028] FIG. 15A-15C: depict exemplary TXNIP and Actin (housekeeping control) protein expression by Western blot analysis of a WT cell line and P4P3F4 clone at day 0, day 3, day 5, day 7 and day 10 of culture.

[0029] FIG. 16A-16C: depict exemplary relative TXNIP protein expression by densitometric analysis of Western blots of a WT cell line (dashed lines) and the P4P3F4 clone (solid lines) at day 0, day 3, day 5, day 7 and day 10 of culture. FIG. 16A culture condition of 4.6 g / L glucose. FIG. 16B culture condition of 1 .2 g / L glucose. FIG. 16C culture condition of HiDOG.DETAILED DESCRIPTION

[0030] Disclosed herein are host cells having improved growth characteristics, and related cell cultures. In some aspects, cells provided herein have increased expression of thioredoxin interacting protein (TXNIP).General Techniques

[0031] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et aL, 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.L Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et aL, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et aL, eds., 1994); Current Protocols in Immunology (J.E. Coligan et aL, eds., 1991 ); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995), as well as in subsequent editions and corresponding websites of the above references, as applicable.Definitions

[0032] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0033] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0034] An "antibody” refers to an immunoglobulin molecule capable of specific binding to a target, such as a polypeptide, carbohydrate, polynucleotide, lipid, etc., through at least one antigen binding site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” can encompass any type of antibody (e.g. monospecific, bispecific, trispecific, etc.), and includes portions of intact antibodies that retain the ability to bind to agiven antigen (e.g. an “antigen-binding fragment”), and any other modified configuration of an immunoglobulin molecule that comprises an antigen binding site. An exemplary antibody comprises i) a variable region of the light chain, heavy chain or both and ii) a constant region of the heavy chain comprising three sequential immunoglobulin domains (CH1 , CH2, and CH3) and of the light chain comprising a single immunoglobulin domain (CL).

[0035] An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or subclass thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains (HC), immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgGs, IgGs, lgG4, IgAi and lgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0036] Examples of antibody antigen-binding fragments and modified configurations include (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CH1 domains); (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); and (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of an Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)); see e.g., Bird et aL, Science 1988; 242:423-426 and Huston et aL, Proc. Natl. Acad. Sci. 1988 USA 85:5879- 5883. Other forms of single chain antibodies, such as diabodies are also encompassed.

[0037] In addition, further encompassed are antibodies that are missing a C-terminal lysine (K) amino acid residue on a heavy chain polypeptide (e.g. human IgG 1 heavy chain comprises a terminal lysine). As is known in the art, the C-terminal lysine is sometimes clipped during antibody production, resulting in an antibody with a heavy chain lacking the C-terminal lysine. Alternatively, an antibody heavy chain may be produced using a nucleic acid that does not include a C-terminal lysine.

[0038] An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding portion thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen binding portion, and any othermodified configuration of the immunoglobulin molecule that comprises an antigen recognition site. Antigen binding portions include, for example, Fab, Fab’, F(ab’)2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), fragments including complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.

[0039] An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or subclass thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG 1 , lgG2, lgG3, lgG4, lgA1 and lgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0040] As used herein, an “exogenous” nucleic acid refers to a recombinant nucleic acid molecule that will be or has been introduced into a host cell (e. g. by conventional genetic engineering methods, preferably by means of transformation, electroporation, lipofection, or transfection), which was prior to said introduction was not present in said host cell. In some circumstances, an exogenous nucleic acid contains a nucleotide sequence that does not naturally occur in the host cell. Such sequences are also termed “transgenic”. In some circumstances, an exogenous nucleic acid may contain a nucleotide sequence of that is the same as a sequence that is endogenous to the cell (e.g. an exogenous nucleic acid molecule may contain a nucleotide sequence of a gene that is endogenous to the host cell, such that introduction of the exogenous nucleic acid molecule into the host cell introduces an additional copy of the gene into the host cell). An “exogenous nucleic acid” refers to an exogenous nucleic acid molecule, or the nucleotide sequence thereof.

[0041] The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” are used interchangeably herein to refer to chains of amino acids of any length. The chain may be linear or branched, it may comprise modified amino acids, and / or may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides can occur as singlechains or associated chains.

[0042] As known in the art, “polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to chains of nucleotides of any length and conformation (e.g. linear or circular) and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5’ and 3’ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2’-O-methyl-, 2’-O-allyl, 2’- fluoro- or 2’-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S(“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR’, CO or CH2 (“formacetal”), in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0043] The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to chains of amino acids of any length. The chain may be linear or branched, it may comprise modified amino acids, and / or may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides can occur as single chains or associated chains.

[0044] As used herein, a “recombinant” nucleic acid refers to a nucleic acid molecule that contains a polynucleotide sequence that does not occur in nature and / or or which is synthetically manufactured. For example, a “recombinant” nucleic acid may contain a proteinencoding gene coupled to a vector sequence. The sequence of the protein-encoding gene may occur in nature, but the gene does not naturally occur in combination with the vector sequence. Put another way, a “recombinant” nucleic acid molecule may contain as part of the molecule a nucleic acid sequence that occurs in nature, but that sequence is either coupled to another sequence (such that the totality of the nucleic acid molecule sequence does not occur in nature) and / or the molecule is synthetically manufactured. A “recombinant” polypeptide refers to a polypeptide produced from a recombinant nucleic acid.

[0045] As used herein, the term “therapeutic molecule” is a molecule that may alleviate or reduce symptoms that result from an absence or defect in a molecule in a target cell (e.g., an isolated cell) or organism (e.g., a subject). In some embodiments, a therapeutic molecule is a therapeutic peptide, polypeptide or protein (e.g., enzyme, structural protein, transmembrane protein, transport protein). A therapeutic polypeptide or protein may be encoded by a recombinant polynucleotide and be expressed by a host cell. In some embodiments, a therapeutic molecule is a recombinant protein encoded by a recombinant polynucleotide and is expressed by a host cell (e.g., a CHO cell). In some embodiments, a recombinant protein is an enzyme, receptor, antibody, hormone, regulatory factor, antigen, binding agent, fusion protein, cytokine, detectable protein.

[0046] As used herein, “vector” means a construct, which is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0047] Where aspects or embodiments of the invention are described in terms of a Markushgroup or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.

[0048] Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting. The term “or” when used in the context of a listing of multiple options (e.g. “A, B, or C”) shall be interpreted to include any one or more of the options, unless the context clearly dictates otherwise. It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of” are also provided.

[0049] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting.Host cells

[0050] As used herein, the term “host cell” or “host cells” means a cell or cells suitable for or adapted to in vitro production of desired biological products. A host cell is often a clonal cell line capable of dividing for multiple generations before senescence stops growth, or may even be immortal. For use in the methods of the disclosure, a host cell can be modified, transiently or non-transiently, through introduction of exogenous genetic information designed to direct biosynthesis in a host cell of specific biological products (e.g., a therapeutic molecule, e.g., an antibody). For example, a host cell can be transfected with a nucleic acid containing a nucleobase sequence encoding a therapeutic molecule, e.g., a protein or regulatory RNA (such as IncRNA, miRNA, or siRNA). In some embodiments, a nucleic acid is DNA, such as a plasmid in which a coding sequence is under the control of a transcriptional regulatory element, such as a promoter and / or enhancer, that can be acted on by the cellular transcription and splicing machinery to produce mRNA. In other embodiments, a nucleic acid can be RNA, such as mRNA, capable of being directly translated into protein.

[0051] Various ways are known in the art for transfecting a host cell with DNA or RNA. These include, without limitation, mixing DNA or RNA with certain compounds that can complex with nucleic acids and then be taken up into a cell, including calcium phosphate or cationicorganic compounds, such as DEAE-dextran, polyethylenimine (PEI), polylysine, polyornithine, polybrene, cyclodextrin, cationic lipids, and others known in the art. Transfection can also be performed non-chemically via electroporation and more exotic technologies, such as biolistic particle delivery. As known in the art, transfection can be transient or stable. With transient transfection, a transfected DNA or RNA exists in a cell for a limited period of time and, in the case of DNA, does not integrate into the genome. With stable transfection, DNA introduced into a cell can persist for long periods either as an episomal plasmid, or integrated into a chromosome. Usually, to produce stably s transfected cell, a plasmid containing a selection marker, as well as a gene or genes for expressing a desired biological product, is transfected into a cell which is then grown and maintained under selective pressure, e.g., conditions that kill non-transfected cells or transfected cells from which the exogenous DNA, including its selection marker, have been lost. For example, plasmids can contain an antibiotic resistance gene and a transfected cell can be selected for by adding an antibiotic to the media in which the cell is grown. In some embodiments, a gene for producing a biological product introduced into a stably transfected host cell is under the control of an inducible promoter which is not expressed, or only at a low level, unless an environmental factor, such as a drug, metal ion, or temperature change, which induces the promoter, is introduced as the cell is grown.

[0052] In some embodiments, a host cell genome can be modified in a non-transient and targeted fashion using genetic engineering methods, such as knock-in, or gene editing methods, to direct a host cell to produce a desired biological product, component thereof, or other gene product necessary for biosynthesis of the biological product. The invention is not limited by the manner in which a host cell is generated. A foreign gene can also be introduced into a host cell for purposes of directing production of a desired biological product by transduction, in which a host cell is infected with modified viruses (e.g., a vector) containing such gene. Examples of viral vectors useful for such purposes include adenovirus, retroviruses (including lentiviruses), baculoviruses, vaccinia virus, and herpes simplex virus, with others being possible.

[0053] A host cell can be any type of cell known in the art to be useful for the purpose of biosynthesizing a desired biological product. A host cell can be a prokaryotic cell, such as bacteria, such as E. coli, or a eukaryotic cell, such as a fungal cell, such as a yeast cell, such as a plant cell, or such as an animal cell, such as an insect cell or mammalian cell, including a rat, mouse, or human cell. In some embodiments, a host cell useful in methods of the disclosure is a mammalian host cell, examples of which include Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980), human cervical carcinoma cells (HeLa, ATCC CCL 2), COS cells (including monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651)), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et aL, J. Gen Virol. , 36:59,1977)(including variants of HEK293 cells, such as HEK293E, HEK293F, HEK293H, HEK293T or HEK293FT cells), A549 cells, baby hamster kidney cells (BHK, ATCC CCL 10), African green monkey kidney cells (VERO-76, ATCC CRL-1 587), NIH 3T3 cells, HT-1080 cells, Sp2 / 0 cells, BALB / c mouse myeloma line (NSO / I, ECACC No: 85110503), C127 cells, AGE1 .HN cells, CAP cells, HKB-11 cells, or human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands), mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 , 1980); monkey kidney cells (CV1 ATCC CCL 70), canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TRI cells (Mather et aL, Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some embodiments, a host cell is a CHO cell. In some embodiments, a host cell is a GS-celL

[0054] For purposes of producing a biological product, a host cell is often grown or maintained in a cell culture under controlled conditions conducive to its growth to relatively high density and biosynthesis of a desired biological product. For example, a host cell can be grown as a cell culture in liquid media of defined chemical composition that provides all the nutrients necessary for cell growth and biosynthesis. Exemplary media includes DMEM, DMEM / F12, MEM and RPMI 1640 for mammalian host cells. Such media may be supplemented with antibiotics, growth factors or cytokines (produced recombinantly or present in animal serum, such as FBS) known to stimulate growth of the particular type of cell in culture, as well as other ingredients that may be required for optimal biosynthesis and / or activity of a desired biological product, but that would otherwise be in limiting supply. Exemplary supplements include essential amino acids, glutamine, vitamin K, insulin, BSA, or transferrin. In addition to growth media, other culture conditions may be controlled to optimize growth and / or productivity of cultured cells, such as pH, temperature and CO2 and oxygen concentration.

[0055] A host cell in culture can be grown or maintained in many containers known in the art, such as a stirred tank bioreactor, wave bag, spinner flask, hollow fiber bioreactor or roller bottle, some of which can be designed and configured for single use or multiple use. Depending on the characteristics of the host cell in question, a host cell can be grown in adherent cell culture, where cells of the culture attach to and grow while in contact with a physical substrate, or in suspension cell culture, either where single cells float free in the media that sustains them, or while attached to bead microcarriers, which are suspended in the media. As known in the art, various technologies have been developed and can be used to grow host cells to high cell density, such as perfusion culture.

[0056] As known in the art, samples of host cells are often maintained in frozen cell banks, such as master cell banks and working cell banks, which facilitate production of biological products in many batches over time, while ensuring consistent performance by the host cells. Before a campaign to produce a biological product, a frozen sample of host cells from a cellbank would typically be thawed, seeded into a small culture volume, and grown to ever higher densities or numbers in cultures of increasing volume. When host cells have reached a desired cell density and / or volume in culture, exogenous genetic material can be introduced, such as by transfection with plasmid DNA or infection with viral vectors, to cause them to begin producing a desired biological product. Or, if using non-transiently modified host cells in which genes for a biological product are under inducible control, the environmental factor necessary to induce expression can be introduced. Host cells can then be grown or maintained in culture for time and under conditions sufficient for them to produce a desired amount of the biological product. Cell Culture Media

[0057] The terms “medium,” “media,” “cell culture medium” and “culture medium” as used herein refer to a solution containing components or nutrients which nourish growing mammalian cells (e.g., host cells). Typically, nutrients include a combination of essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and / or survival. Such a solution may also contain a further nutrient or supplementary component that enhances growth and / or survival above the minimal rate, including, but not limited to, a hormone and / or another growth factor, an ion (such as sodium, chloride, calcium, magnesium, and phosphate), a buffer, a vitamin, a nucleoside or nucleotide, a trace element (inorganic compounds usually present at very low final concentrations), an inorganic compound present at a high final concentrations(e.g., iron), an amino acid, lipid, glucose and / or, another energy source, or a combination thereof. In some embodiments, a medium is advantageously formulated to a pH and salt concentration optimal for cell survival and proliferation. In some embodiments, a medium is a feed medium that is added after the beginning of the cell culture.

[0058] A wide variety of mammalian cell (e.g., host cell) growth media may be used in accordance with the present invention. In some embodiments, a cell may be grown in one of a variety of chemically defined media, wherein components of the media are both known and controlled. In some embodiments, a cell may be grown in a complex medium, in which not all components of the medium are known and / or controlled.

[0059] Chemically defined growth media for mammalian cell culture (e.g., host cell culture) have been extensively developed and published. All components of defined media are well characterized, and so defined media do not contain complex additives such as serum or hydrolysates. In some embodiments, media formulations have been developed with the express purpose of supporting highly productive recombinant protein producing cell cultures. Such media generally comprises high amounts of nutrients and in particular high amounts of amino acids to support growth and / or maintenance of host cells at high density.

[0060] Not all components of complex media are well characterized, and so complex mediamay contain additives such as simple and / or complex carbon sources, simple and / or complex nitrogen sources, and serum, among other additives. In some embodiments, complex media suitable for cells (e.g., host cells, e.g., CHO cells) and methods of the present disclosure contain additives such as hydrolysates in addition to other components of defined medium as described herein.

[0061] In some embodiments, a defined media includes chemical entities or components at known concentrations in water. Some media formulations contain one or more well- characterized proteins such as insulin, IGF-1 , transferrin or BSA, whereas others require no protein components and so are referred to as protein-free defined media. Typical chemical components of the media fall into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and a miscellaneous category (e.g., a supplementary component).

[0062] Cell culture medium may be optionally supplemented with a supplementary component. The term “supplementary component” as used herein refers to a component that enhances growth and / or survival above the minimal rate, including, but not limited to, a hormone and / or other growth factor, an ion (such as sodium, chloride, calcium, magnesium, and phosphate), a buffer, a vitamin, a nucleoside or nucleotide, a trace element (an inorganic compound usually present at a very low final concentration), an amino acid, lipid, glucose, and / or other energy source, or a combination thereof. In some embodiments, a supplementary component may be added to the initial cell culture. In some embodiments, a supplementary component may be added after the beginning of the cell culture.

[0063] A component which is a trace element refers to a variety of inorganic salts included at micromolar or lower levels. For example, a commonly included trace element is zinc, selenium, copper, and others. In some embodiments, iron (ferrous or ferric salts) can be included as a trace element in the initial cell culture medium at micromolar concentrations. Manganese is also frequently included among the trace elements as a divalent cation (MnCh or MnSC ) in a range of nanomolar to micromolar concentrations. Numerous less common trace elements are usually added at nanomolar concentrations.

[0064] In some embodiments, a medium used in methods of the disclosure is a medium suitable for supporting high cell density, such as for example 1 x 106cells per milliliter cell culture media (cells / mL), 5 x 106cells / mL, 1 x 107cells / mL, 5 x 107cells / mL, 1X108cells / mL or 5X108cells / mL, in a cell culture. In some embodiments, a cell culture is a mammalian cell fed- batch culture, optionally a CHO cell fed-batch culture.

[0065] In some embodiments, cell culture medium is HiPDOG medium, medium used in the HipDOG process, but comprising no tyrosine. In some embodiments, cell culture medium comprises no tyrosine wherein a HiPDOG culture or process is used which comprises no tyrosine. HiPDOG culture conditions are disclosed, e.g., in W02004 / 104186 which isincorporated herein by refence.Cells Having Increased TXNIP

[0066] In some embodiments, provided herein are mammalian host cells having at least one of 1 ) overexpression of the thioredoxin interacting protein (TXNIP) gene and 2) increased copy number of the TXNIP gene. In some embodiments, a host cell with an increased copy number of the TXNIP gene comprises an exogenous TXNIP gene.

[0067] As used herein, the term “TXNIP” includes variants, isoforms, homologs, orthologs and paralogs of TXNIP, and includes TXNIP from species known to persons of skill in the art, including mouse, rat, Chinese Hamster, and humans.

[0068] In one example, the TXNIP gene is mouse (Mus musculus) TXNIP.

[0069] The amino acid sequence for the mouse TXNIP polypeptide is available underGen Bank accession number AAH31850.1 and is shown here:

[0070] MVMFKKIKSFEVVFNDPEKVYGSGEKVAGRVIVEVCEVTRVKAVRILACGVAKVLWMQGSQQCKQTLDYLRYEDTLLLEEQPTGENEMVIMRPGNKYEYKFGFELPQGPLGTSFKGKYGCVDYWVKAFLDRP SQPTQEAKKNFEVMDLVDVNTPDLMAPVSAKKEKKVSCMFIPDGRVSVSARIDRKGFCEGDDISIHADFE NTCSRIWPKAAIVARHTYLANGQTKVFTQKLSSVRGNHI ISGTCASWRGKSLRVQKIRPSILGCNILKV EYSLLIYVSVPGSKKVILDLPLVIGSRSGLSSRTSSMASRTSSEMSWIDLNIPDTPEAPPCYMDI IPEDH RLESPTTPLLDDVDDSQDSP IFMYAPEFQFMPPPTYTEVDPCVLNNNNNNNNNVQ (SEQ ID NO: 1)

[0071] The polynucleotide sequence for the mouse TXNIP gene is available under GenBank accession number BC031850.1 , and comprises the polynucleotide sequence:

[0072] ATGGTGATGTTCAAGAAGATCAAGTCTTTTGAGGTGGTCTTCAACGACCCCGAGAAGGTGTA CGGCAGCGGGGAGAAGGTGGCCGGACGGGTGATAGTGGAAGTGTGTGAAGTTACCCGAGTCAAAGCCGTC AGGATCCTGGCTTGCGGCGTGGCCAAGGTCCTGTGGATGCAAGGGTCTCAGCAGTGCAAACAGACTTTGG ACTACTTGCGCTATGAAGACACACTTCTCCTAGAAGAGCAGCCTACAGGTGAGAACGAGATGGTGATCAT GAGGCCTGGAAACAAATATGAGTACAAGTTCGGCTTCGAGCTTCCTCAAGGGCCCCTGGGAACATCCTTT AAAGGAAAATATGGTTGCGTAGACTACTGGGTGAAGGCTTTTCTCGATCGCCCCAGCCAGCCAACTCAAG AGGCAAAGAAAAACTTCGAAGTGATGGATCTAGTGGATGTCAATACCCCTGACTTAATGGCACCAGTGTC TGCCAAAAAGGAGAAGAAAGTTTCCTGCATGTTCATTCCTGATGGACGTGTGTCAGTCTCTGCTCGAATT GACAGAAAAGGATTCTGTGAAGGTGATGACATCTCCATCCATGCCGACTTTGAGAACACGTGTTCCCGAA TCGTGGTCCCCAAAGCGGCTATTGTGGCCCGACACACTTACCTTGCCAATGGCCAGACCAAAGTGTTCAC TCAGAAGCTGTCCTCGGTCAGAGGCAATCACATTATCTCAGGGACTTGCGCATCGTGGCGTGGCAAGAGC CTCAGAGTGCAGAAGATCAGACCATCCATCCTGGGCTGCAACATCCTCAAAGTCGAATACTCCTTGCTGA TCTACGTCAGTGTCCCTGGCTCCAAGAAAGTCATCCTTGATCTGCCCCTAGTGATTGGCAGCAGGTCGGG TCTGAGCAGCCGGACATCCAGCATGGCCAGCCGGACGAGCTCTGAGATGAGCTGGATAGACCTAAACATC CCAGATACCCCAGAAGCTCCTCCTTGCTATATGGACATCATTCCTGAAGATCACAGACTAGAGAGCCCCA CCACCCCTCTGCTGGACGATGTGGACGACTCTCAAGACAGCCCTATCTTTATGTACGCCCCTGAGTTCCAGTTCATGCCCCCACCCACTTACACTGAGGTGGATCCGTGCGTCCTTAACAACAACAACAACAACAACAAC AACGTGCAGTGA (SEQ ID NO: 2).

[0073] In some embodiments, provided herein are mammalian host cells having overexpression of the thioredoxin interacting protein (TXNIP) gene.

[0074] In some embodiments, “overexpression” refers to expression of a TXNIP gene that is increased as compared to expression of a TXNIP gene in an otherwise identical cell. In some embodiments, overexpression of a TXNIP gene in a cell (e.g., a mammalian host cell) is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold greater than expression of a TXNIP gene by an otherwise identical cell.

[0075] In some embodiments, “overexpression” refers to expression of a TXNIP polypeptide that is increased as compared to expression of a TXNIP polypeptide in an otherwise identical cell. In some embodiments, overexpression of a TXNIP polypeptide in a cell (e.g., a mammalian host cell) is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of a TXNIP polypeptide by an otherwise identical cell.

[0076] In some embodiments, a TXNIP gene in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with a molecule that increases transcription of TXNIP mRNA as compared to TXNIP gene expression by an otherwise identical cell that is not contacted with the molecule that increases transcription of TXNIP mRNA. In some embodiments, overexpression of a TXNIP gene in a cell (e.g., a mammalian host cell) that is contacted with a molecule that increases transcription of a TXNIP mRNA is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of a TXNIP gene by an otherwise identical cell that is not contacted with the molecule that increases transcription of TXNIP mRNA.

[0077] In some embodiments, a TXNIP polypeptide in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with a molecule that increases translation of the TXNIP polypeptide as compared to TXNIP polypeptide expression by an otherwise identical cellthat is not contacted with the molecule that increases translation of the TXNIP polypeptide. n some embodiments, overexpression of a TXNIP polypeptide in a cell (e.g., a mammalian host cell) that is contacted with a molecule that increases translation of the TXNIP polypeptide is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold greater than expression of a TXNIP polypeptide by an otherwise identical cell that is not contacted with the molecule that increases translation of the TXNIP polypeptide.

[0078] In some embodiments, a TXNIP gene in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with glucosamine. In some embodiments, a TXNIP gene in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with glucosamine as compared to TXNIP gene expression by an otherwise identical cell that is not contacted with glucosamine. In some embodiments, overexpression of a TXNIP gene in a cell (e.g., a mammalian host cell) that is contacted with a glucosamine is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of a TXNIP gene by an otherwise identical cell that is not contacted with glucosamine.

[0079] In some embodiments, a TXNIP polypeptide in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with glucosamine. In some embodiments, a TXNIP polypeptide in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with glucosamine as compared to TXNIP polypeptide expression by an otherwise identical cell that is not contacted with glucosamine. In some embodiments, overexpression of a TXNIP polypeptide in a cell (e.g., a mammalian host cell) that is contacted with a glucosamine is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold greater than expression of a TXNIP polypeptide by an otherwise identical cell that is not contacted with glucosamine.

[0080] In some embodiments, a TXNIP gene in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule, and the second molecule is an inhibitor of TXNIP gene expression. In some embodiments, a TXNIP gene in a cell (e.g., a mammalian host cell)is overexpressed when the cell is contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule, and the second molecule is an inhibitor of TXNIP gene expression, as compared to TXNIP gene expression by an otherwise identical cell that is not contacted with the first molecule.

[0081] In some embodiments, overexpression of a TXNIP gene in a cell (e.g., a mammalian host cell) that is contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule, and the second molecule is an inhibitor of TXNIP gene expression, is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold greater than the expression of a TXNIP gene by an otherwise identical cell that is not contacted with the first molecule.

[0082] In some embodiments, a TXNIP polypeptide in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule, and the second molecule is an inhibitor of TXNIP polypeptide expression. In some embodiments, a TXNIP polypeptide in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule, and the second molecule is an inhibitor of TXNIP polypeptide expression, as compared to TXNIP polypeptide expression by an otherwise identical cell that is not contacted with the first molecule.

[0083] In some embodiments, overexpression of a TXNIP polypeptide in a cell (e.g., a mammalian host cell) that is contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule, and the second molecule is an inhibitor of TXNIP polypeptide expression, is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of a TXNIP polypeptide by an otherwise identical cell that is not contacted with the first molecule.

[0084] In some embodiments, a molecule that inhibits, reduces or decreases TXNIP gene expression is a MYC polypeptide. In some embodiments, a molecule that inhibits, reduces or decreases TXNIP polypeptide expression is a MYC polypeptide.

[0085] The MYC polypeptide is an oncogene transcription factor with a wide array of functions affecting cellular activities such as cell cycle, apoptosis, DNA damage response and hematopoiesis (Genebank Ref No. NG_007161 ; UniProt No. P01106). MYC expression isregulated at multiple levels in a cell. A MYC polypeptide may inhibit TXNIP gene expression or TXNIP polypeptide expression in a cell (e.g., a mammalian host cell). Conversely, when expression of a MYC gene, polypeptide or both in a cell (e.g., a mammalian host cell) is inhibited or reduced, increased expression, or overexpression, of a TXNIP gene, polypeptide or both may occur.

[0086] MYC gene expression, protein expression, activity or a combination thereof may be inhibited by a small molecule, for example, JQ1 , OTX-015, MYCM1 -6, EN4, KJPyr9, APTO- 253, 10058-F4, Jy-3-094, MYCi361 and a combination thereof. A molecule that inhibits MYC activity may do so by, for example, inhibiting MYC gene expression, inhibiting MYC protein expression, degrading a MYC polypeptide, blocking MYC heterodimerization and blocking MYC protein interactions.

[0087] In some embodiments, a TXNIP gene in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with a molecule that inhibits MYC activity, as compared to TXNIP gene expression by an otherwise identical cell that is not contacted with the molecule that inhibits MYC activity. In some embodiments, a molecule that inhibits MYC activity is JQ1 or OTX-015. In some embodiments, overexpression of a TXNIP gene in a cell (e.g., a mammalian host cell) that is contacted with a molecule that inhibits MYC activity is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold greater than expression of a TXNIP gene by an otherwise identical cell that is not contacted with the molecule that inhibits MYC activity. In some embodiments, a molecule that inhibits MYC activity is JQ1 or OTX-015.

[0088] In some embodiments, a TXNIP polypeptide in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with a molecule that inhibits MYC activity, as compared to TXNIP polypeptide expression by an otherwise identical cell that is not contacted with the molecule that inhibits MYC activity. In some embodiments, a molecule that inhibits MYC activity is JQ1 or OTX-015. In some embodiments, overexpression of a TXNIP polypeptide in a cell (e.g., a mammalian host cell) that is contacted with a molecule that inhibits MYC activity is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of a TXNIP polypeptide by an otherwise identical cell that is not contacted with the molecule that inhibits MYC activity. In some embodiments, a molecule that inhibits MYC activity is JQ1 or OTX-015.

[0089] In some embodiments, a TXNIP gene in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with a first molecule that inhibits MYC activity and a second molecule that increases expression of the TXNIP gene, as compared to TXNIP gene expression by an otherwise identical cell that is not contacted with the first molecule and the second molecule. In some embodiments, a molecule that inhibits MYC activity is JQ1 or OTX- 015. In some embodiments, a molecule that increases expression of a TXNIP gene is glucosamine.

[0090] In some embodiments, overexpression of a TXNIP gene in a cell (e.g., a mammalian host cell) that is contacted with a first molecule that inhibits MYC activity and a second molecule that increases expression of the TXNIP gene is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of a TXNIP gene by an otherwise identical cell that is not contacted with the first molecule and the second molecule. In some embodiments, a molecule that inhibits MYC activity is JQ1 or OTX-015. In some embodiments, a molecule that increases expression of a TXNIP gene is glucosamine.

[0091] In some embodiments, a TXNIP polypeptide in a cell (e.g., a mammalian host cell) is overexpressed when the cell is contacted with a first molecule that inhibits MYC activity and a second molecule that increases expression of the TXNIP polypeptide, as compared to TXNIP polypeptide expression by an otherwise identical cell that is not contacted with the first molecule and the second molecule. In some embodiments, a molecule that inhibits MYC activity is JQ1 or OTX-015. In some embodiments, a molecule that increases expression of a TXNIP polypeptide is glucosamine.

[0092] In some embodiments, overexpression of a TXNIP polypeptide in a cell (e.g., a mammalian host cell) that is contacted with a first molecule that inhibits MYC activity and a second molecule that increases expression of a TXNIP polypeptide is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of a TXNIP polypeptide by an otherwise identical cell that is not contacted with the first molecule and the second molecule.

[0093] In some embodiments, a cell (e.g., a mammalian host cell) comprises an exogenous TXNIP gene. In some embodiments, a nucleic acid sequence encoding a TXNIP polypeptideencodes a polypeptide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1 .

[0094] In some embodiments, a nucleic acid sequence encoding a TXNIP polypeptide comprises a polynucleotide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:2.

[0095] In some embodiments, a cell (e.g., a mammalian host cell) comprises an exogenous TXNIP gene and the nucleic acid sequence of the TXNIP gene is chromosomally-integrated into a host cell chromosome.

[0096] In some embodiments, a cell chromosome contains a recombination target site for site-specific integration of an exogenous nucleic acid sequence encoding TXNIP into the host cell chromosome.

[0097] In some embodiments, a cell (e.g., a mammalian host cell, e.g. ,a CHO cell) comprises an exogenous TXNIP gene and overexpresses TXNIP mRNA, overexpresses TXNIP polypeptide, has increased TXNIP activity or a combination thereof. In some embodiments, an exogenous TXNIP gene is a mouse gene.

[0098] In some embodiments, a CHO cell comprises an exogenous TXNIP gene.

[0099] In some embodiments, a CHO cell comprising an exogenous TXNIP gene overexpresses TXNIP mRNA, overexpresses TXNIP polypeptide, has increased activity or a combination thereof. In some embodiments, an exogenous TXNIP gene is a mouse gene.

[0100] In some embodiments, a CHO cell comprises an exogenous TXNIP and an exogenous gene encoding a therapeutic molecule, or a portion thereof.

[0101] In some embodiments, a cell comprises an exogenous TXNIP gene and an exogenous gene encoding a therapeutic molecule or a portion thereof, wherein the nucleic acid sequence of the TXNIP gene, the nucleic acid sequence encoding the therapeutic molecule or portion thereof, or both, is chromosomally-integrated into a host cell chromosome.

[0102] In some embodiments, a therapeutic molecule is a recombinant protein, for example an enzyme, receptor, antibody, hormone, regulatory factor, antigen, binding agent, fusion protein, cytokine, detectable protein (e.g., a fluorescent protein such as a green fluorescent protein).

[0103] In some embodiments, a cell (e.g., a mammalian host cell, e.g., a CHO cell) comprises an exogenous TXNIP gene and overexpresses the TXNIP gene, overexpresses a TXNIP polypeptide, has increased TXNIP activity, or a combination thereof, as compared to expression of a TXNIP gene, expression of a TXNIP polypeptide, TXNIP activity or a combination thereof by an otherwise identical cell that does not comprise an exogenous TXNIP gene.

[0104] In some embodiments, a cell comprising an exogenous TXNIP gene overexpressesthe TXNIP gene, overexpresses a TXNIP polypeptide, has increased TXNIP activity or a combination thereof, by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to expression of a TXNIP gene, expression of a TXNIP polypeptide, TXNIP activity or a combination thereof, by an otherwise identical cell that does not comprise an exogenous TXNIP gene.

[0105] In some embodiments, a cell (e.g., a mammalian host cell, e.g., a CHO cell) comprises an exogenous TXNIP gene and an exogenous gene encoding a therapeutic molecule or a portion thereof and overexpresses the therapeutic molecule or portion thereof as compared to an otherwise identical cell that does not comprise an exogenous TXNIP gene.

[0106] In some embodiments, a cell comprising an exogenous TXNIP gene and an exogenous gene encoding a therapeutic molecule or a portion thereof overexpresses the therapeutic molecule or portion thereof, by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to expression of the therapeutic molecule or portion thereof by an otherwise identical cell that does not comprise an exogenous TXNIP gene.

[0107] In some embodiments, a cell culture comprising a cell comprising an exogenous TXNIP gene and an exogenous gene encoding a therapeutic antibody (e.g., an IgG antibody) produces at least 0.01 grams antibody per liter cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1 .2 g / L, 1 .4 g / L, 1 .6 g / L, 1 .8 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L. 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 / g / L or 20 g / L.

[0108] In some embodiments, a cell (e.g., a mammalian host cell, e.g., a CHO cell) comprises an exogenous TXNIP gene and i) has reduced synthesis of a growth inhibitor, ii) has reduced synthesis of a productivity inhibitor, iii) has an improved growth characteristics or iv) a combination thereof, as compared to an otherwise identical cell that does not comprise an exogenous TXNIP gene.

[0109] In some embodiments, a cell comprising an exogenous TXNIP gene has reduced synthesis of a growth inhibitor as compared to an otherwise identical cell that does not comprise an exogenous TXNIP gene.

[0110] In some embodiments, a cell that overexpresses a TXNIP gene has reducedsynthesis of a growth inhibitor as compared to an otherwise identical cell that does not overexpress a TXNIP gene.

[0111] In some embodiments, a cell that overexpresses a TXNIP polypeptide has reduced synthesis of a growth inhibitor as compared to an otherwise identical cell that does not overexpress a TXNIP polypeptide.

[0112] In some embodiments, synthesis of a growth inhibitor by a cell that comprises an exogenous TXNIP gene, overexpresses a TXNIP gene, overexpresses a TXNIP polypeptide, or a combination thereof, is reduced by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to synthesis of a growth inhibitor by an otherwise identical cell that does not comprise an exogenous TXNIP gene, does not overexpress a TXNIP gene, does not overexpress a TXNIP polypeptide, or a combination thereof.

[0113] As used herein “growth inhibitor” refers to any molecule produced by a cell in culture that reduces, inhibits or decreases the growth rate of the cells in the culture. A growth inhibitor may also decrease the viability of the cells in the culture such that the total number of viable cells in the culture does not increase, or remains constant, over time.

[0114] Growth inhibitors may include cell metabolites. Cell metabolites may result from metabolism of amino acids, carbohydrates (e.g., glucose), fatty acids, or a combination thereof. Cell metabolites may result from metabolism of amino acids including phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, glycine or a combination thereof. Cell metabolites of phenylalanine and tyrosine metabolism include 3-(4-hydroxyphenyl)lactate, 4- hydroxyphenylpyruvate, or both. A cell metabolite of phenylalanine metabolism is phenyllactate. Cell metabolites of tryptophan metabolism include indolelactate (indole-3- lactate), indolecarboxylic acid (indole-3-carboxylic acid) or both. Cell metabolites of methionine metabolism include homocysteine, 2-hydroxybutyric acid, or both. A cell metabolite of leucine metabolism is isovalerate. A cell metabolite of serine, threonine and glycine metabolism is formate.

[0115] In some embodiments, a growth inhibitor is selected from the group consisting of 3- (4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate (indole-3- lactate), indolecarboxylic acid (indole-3-carboxylic acid), homocysteine, 2-hydroxybutyric acid, isovalerate, butyrate, isobutyrate, 2-methylbutyrate, formate, lactate and a combination thereof.

[0116] In some embodiments, a growth inhibitor is lactate. In some embodiments, a growth inhibitor is ammonia.

[0117] In some embodiments, a cell comprising an exogenous TXNIP gene has reduced synthesis of a productivity inhibitor as compared to an otherwise identical cell that does not comprise an exogenous TXNIP gene.

[0118] In some embodiments, a cell that overexpresses a TXNIP gene has reduced synthesis of a productivity inhibitor as compared to an otherwise identical cell that does not over express a TXNIP gene.

[0119] In some embodiments, a cell that overexpresses a TXNIP polypeptide has reduced synthesis of a productivity inhibitor as compared to an otherwise identical cell that does not overexpress a TXNIP polypeptide.

[0120] In some embodiments, a cell that has increased TXNIP activity has reduced synthesis of a productivity inhibitor as compared to an otherwise identical cell that does not have increased TXNIP activity.

[0121] In some embodiments, synthesis of a productivity inhibitor by a cell that comprises an exogenous TXNIP gene, overexpresses a TXNIP gene, overexpresses a TXNIP polypeptide, has increased TXNIP activity or a combination thereof, is reduced by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to synthesis of a productivity inhibitor by an otherwise identical cell that does not comprise an exogenous TXNIP gene, does not overexpress a TXNIP gene, does not overexpress a TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.

[0122] As used herein, “productivity inhibitor” refers to any molecule produced by a cell in culture that reduces, inhibits or decreases the production of a therapeutic molecule (e.g., a recombinant protein, e.g., an antibody) that is produced by the cells in the culture. In some embodiments, a growth inhibitor may also act as a productivity inhibitor by reducing, inhibiting or decreasing the growth rate or viability of cells in culture such that the effect is to reduce, inhibit or decrease production of a therapeutic molecule that is produced by the cells in the culture.

[0123] Productivity inhibitors may include cell metabolites. Cell metabolites may result from metabolism of amino acids, carbohydrates (e.g., glucose), fatty acids, or a combination thereof. Cell metabolites may result from metabolism of amino acids including phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, glycine or a combination thereof. Cell metabolites of phenylalanine and tyrosine metabolism include 3-(4-hydroxyphenyl)lactate, 4- hydroxyphenylpyruvate, or both. A cell metabolite of phenylalanine metabolism is phenyllactate.Cell metabolites of tryptophan metabolism include indolelactate (indole-3-lactate), indolecarboxylic acid (indole-3-carboxylic acid) or both. Cell metabolites of methionine metabolism include homocysteine, 2-hydroxybutyric acid, or both. A cell metabolite of leucine metabolism is isovalerate. A cell metabolite of serine, threonine and glycine metabolism is formate.

[0124] In some embodiments, a productivity inhibitor is selected from the group consisting of 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate (indole-3- lactate), indolecarboxylic acid (indole-3-carboxylic acid), homocysteine, 2-hydroxybutyric acid, isovalerate, butyrate, isobutyrate, 2-methylbutyrate, formate, lactate and a combination thereof.

[0125] In some embodiments, a productivity inhibitor is lactate. In some embodiments, a productivity inhibitor is ammonia.

[0126] A productivity inhibitor may decrease production of a therapeutic molecule by cells in culture by reducing, inhibiting or decreasing the transcription of a gene present in a cell in culture that encodes the therapeutic molecule. In some embodiments, a gene encoding a therapeutic molecule present in a cell in culture is an exogenous gene. In some embodiments, a gene encoding a therapeutic molecule in a cell in culture encodes a recombinant protein (e.g., antibody, a fusion protein).

[0127] A productivity inhibitor may decrease production of a therapeutic molecule by cells in culture by reducing, inhibiting or decreasing the translation of mRNA in a cell in culture that encodes the therapeutic molecule. In some embodiments, an mRNA encoding a therapeutic molecule is transcribed from an exogenous gene encoding a therapeutic molecule present in a cell in culture. In some embodiments, a gene encoding a therapeutic molecule in a cell in culture encodes a recombinant protein (e.g., antibody, a fusion protein).

[0128] Identification of a cell metabolite (e.g., a productivity inhibitor, growth inhibitor) can be performed using methods for identifying and / or measuring the metabolite concentration in the cell culture medium or in cells, for example in a cell pellet. A cell metabolite can be identified using one or more of nuclear magnetic resonance (NMR), liquid chromatography with mass spectrometry (LC / MS) and gas chromatography with mass spectrometry (GC / MS) techniques. For metabolomic analysis, a spent medium sample and / or a cell pellet sample can be collected and analyzed, for example, from single or from duplicate reactor runs, performed for each condition taken at time points throughout culture, for example up to and / or including maximum viable cell density, for example, at time points of 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 8 days, 9 days, 11 days, and 12 days. A relative level (fold change) of a metabolite can be measured and calculated. A relative level of a metabolite can be determined in a spent medium sample and / or a cell pellet sample, and may be calculated based on a fold change compared to the level of the metabolite when first detected until the desired timeendpoint, usually maximum viable cell density.

[0129] Quantifying a cell metabolite production, concentration or both can be performed using methods for identifying and / or measuring the metabolite concentration in a cell culture medium or in cells (e.g., a cell pellet sample). In some embodiments, measurement of and / or the quantification of a metabolite is performed using LC / MS, GC / MS, NMR or a combination thereof. In some embodiments, a cell metabolites is identified from a sample of the culture medium or from the cultured cells during cell culture.

[0130] Measurement of and / or quantification of a cell metabolite can be carried out using purified compound forms of the metabolite. A purified compound can be used to prepare a calibration curve at a known concentration in order to precisely correlate measurements obtained from methods used with the concentration of a metabolite. In this way, a precise measurement of the concentration of the metabolite is determinable at any given time point in the cell culture, for example at maximum viable cell density.

[0131] In some embodiments, a viable cell density is at least 5 x 105cells / mL, 10 x 105cells / mL, 25 x 105cells / mL, 50 x 105cells / mL, 100 x 105cells / mL, 150 x 105cells / mL, 200 x 105cells / mL, 250 x 105cells / mL, 300 x 105cells / mL, 350 x 105cells / mL, 400 x 105cells / mL, 450 x 5005cells / mL, 550 x 105cells / mL, 600 x 105cells / mL or 10 x 6505cells / mL.

[0132] The effect of a quantified concentration of a cell metabolite on cell growth and / or productivity of cells in cell culture can be determined by introduction of a known concentration into a cell culture and comparing the effect on cell growth and / or productivity with an otherwise identical culture lacking the introduced cell metabolite.

[0133] In one aspect, provided herein are mammalian host cells (e.g., CHO cells) having one or more improved growth characteristics. In some embodiments, a cell comprising an exogenous TXNIP gene has an improved growth characteristic as compared to an otherwise identical cell that does not comprise an exogenous TXNIP gene. In some embodiments, a cell that overexpresses a TXNIP gene has an improved growth characteristic as compared to an otherwise identical cell that does not overexpress a TXNIP gene. In some embodiments, a cell that overexpresses a TXNIP polypeptide has an improved growth characteristic as compared to an otherwise identical cell that does not overexpress a TXNIP polypeptide. In some embodiments, a cell that has increased TXNIP activity has an improved growth characteristic as compared to an otherwise identical cell that does not overexpress a TXNIP polypeptide.

[0134] In some embodiments, a growth characteristic of a cell that comprises an exogenous TXNIP gene, overexpresses a TXNIP gene, overexpresses a TXNIP polypeptide, has increased TXNIP activity or a combination thereof, is improved by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to a growth characteristic of an otherwise identical cell that does not comprise an exogenous TXNIP gene, does not overexpress a TXNIP gene, does not overexpress a TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.

[0135] As used herein, “growth characteristic” refers to a feature of a cell or cell culture that is correlated with cell growth. Cell growth may be assessed by measuring, for example, 1) cell count (also known as “cell number”), 2) cell viability, 3) viable cell count, 4) cell density, 5) viable cell density, 6) cell metabolism, 7) cell size, or a combination thereof. Frequently, multiple aspects of a cell culture are examined at the same time when assessing cell growth. For example, cultured cells are commonly assessed for “viable cell density”, which takes into account both the number of cells and viability of cells in a cell culture. In addition, cell growth may be assessed by measuring any of the above parameters over a period of time. For example, cell growth can be assessed by measuring the time it takes for the number of cells in a culture to double in number (i.e. “doubling time”). Cells may be counted manually (e.g. with a hemocytometer), or via an automated instrument. Automated cell counter instruments include, for example, Cedex HiRes System (Roche), LUNA (Logos Biosystem), Nova FLEX analyzer (Nova Biomedical), CELLOMETER Auto T4Cell Viability Counter (Peqlab), TC10 and TC20 (Bio-Rad), COUNTESS Automated Cell Counter (Invitrogen), and VI-CELL Cell Viability Analyzer (Beckman Coulter). Cells can also be counted in automated bioreactor systems (e.g. AMBR (Sartorius)) that contain an integrated cell counter. Collectively, automated cell counter and bioreactor systems may be referred to herein as “cell analyzers”. In some embodiments, when cells are counted, the amount or concentration of cells is expressed as the number of cells per mL of media (e.g., 20 cells / mL, 30 cells / mL, 40 cells / mL, 50 cells / mL, 60 cells / mL, 70 cells / mL, 80 cells / mL, 90 cells / mL, 100 cells / mL, 120 cells / mL, 140 cells / mL, 160 cells / mL, 180 cells / mL, 200 cells / mL or more).

[0136] A viable cell can be identified by various methods known in the art. For example, a viable cell can be identified by exposing a cell to a dye that selectively binds either living or dead cells. Dyes that stain a cell that is dead but does not stain a cell that is living include trypan blue, eosin, and propidium. The membrane of a living cell excludes these dyes, but a dead cell membrane do not exclude the dyes. Additional dyes that can be used to assess cell viability include, for example, dyes that bind to DNA (e.g. ethidium monoazide) or to phosphatidylserine (e.g., Annexin V).

[0137] Cell viability can also be sampled via assays in which a living cell converts a substrate to a product that can be readily detected (such as a colored or fluorescent product). In these assays, the amount of detectable product generated is proportional to the viability of the assayed cell. Representative assays include, for example tetrazolium reduction assays (e.g.CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega); Cell Growth Determination Kit (Sigma-Aldrich); MTT Cell Growth Assay Kit (Millipore); CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega); In Vitro Toxicology Assay Kit, XTT (Sigma-Aldrich); Cell Counting Kit-8, WST-8 based (Dojindo Molecular Technologies)), resazurin reduction assays (e.g. CELLTITER-BLUE Cell Viability Assay (Promega); In Vitro Toxicology Assay Kit, Resazurin (Sigma-Aldrich)), protease substrate assays (e.g. using substrate glycylphenylalanyl- aminofluourocoumarin (GF-AFC); CELLTITER-FLUOR Cell Viability Assay (Promega)); ATP I luciferase assays (e.g. CELLTITER-GLO Luminescent Cell Viability Assay (Promega); ATPLITE 1 Step (Perkin Elmer); ATP Bioluminescent Cell Assay Kit (Sigma-Aldrich)).

[0138] Cell metabolism can examine, for example, DNA synthesis in a cell (e.g. BrdU assay or EdU assay) or nuclear proteins associated with cellular proliferation (e.g. anti-Ki67 antibodies).

[0139] Multiple aspects of cell cultures can be examined simultaneously. For example, in some embodiments, cell analyzers can determine multiple parameters including the number of cells and cell viability (i.e. to determine the number of viable cells). For example, the automated VI-CELL Cell Viability Analyzer (Beckman Coulter) uses trypan blue staining for identification of viable cells, and provides automated measurements of, for example, cell number, cell size, percent viability, total cell density, and viable cell density. In another example, the AMBR (Sartorius) bioreactor system can combine a bioreactor for culturing multiple cell cultures with sensors and a cell analyzer for evaluating multiple features of the cell cultures, including cell count, cell viability, and metabolites (lactate, glucose, etc.). In another example, the Cell Viability Imaging kit (Sigma) includes reagents for simultaneous staining of viable, dead, and total cells in a sample, using calcein-AM, propidium iodide, and Hoeschst 33342 dyes, respectively.

[0140] A cell or cell culture that has an “improved growth characteristic” (or the like) as compared to a reference cell or cell culture will have a greater value (or, where appropriate, a smaller value, where the smaller value indicates faster growth) for at least one, two, three, four or more of the above characteristics (e.g., cell number, cell viability, cell metabolism, cell size, etc.) than the reference cell or cell culture over which it has an “improved growth characteristic”.

[0141] A cell with an “improved growth characteristic” refers to a cell that when cultured, produces a first cell culture with improvement of at least one of 1) cell count, 2) cell viability, 3) viable cell count, 4) cell density, 5) viable cell density, 6) cell metabolism, 7) cell size, or a combination thereof that is greater than at least one 1) cell count, 2) cell viability, 3) viable cell count, 4) cell density, 5) viable cell density, 6) cell metabolism and 7) cell size, as compared to a second cell culture that is grown under the same conditions, and for the same period of time, and comprises an otherwise identical cell that does not have the improved growthcharacteristic.

[0142] A cell with an “improved growth characteristic” refers to a cell that when cultured, produces a first cell culture with a 1 ) cell count, 2) cell viability, 3) viable cell count, 4) cell density, 5) viable cell density, 6) cell metabolism, 7) cell size, or a combination thereof that is at least about 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% greater than at least one 1 ) cell count, 2) cell viability, 3) viable cell count, 4) cell density, 5) viable cell density, 6) cell metabolism, 7) cell size or combination thereof, as compared to a second cell culture that is grown under the same conditions, and for the same period of time, and comprises an otherwise identical cell that does not have the improved growth characteristic.

[0143] In some embodiments, a cell or cell culture that has an “improved growth characteristic” as compared to a reference cell or cell culture will have a total cell count or a viable cell count which is at least about 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% greater than the total cell count or viable cell count of a reference cell or cell culture, when the cells are cultured under the same conditions and for the same period of time.

[0144] In some embodiments, a cell or cell culture that has an “improved growth characteristic” as compared to a reference cell or cell culture will have a total cell density or a viable cell density which is at least about 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% greater than the total cell density or viable cell density of a reference cell or cell culture, when the cells are cultured under the same conditions and for the same period of time.

[0145] In some embodiments, a cell or cell culture that has an “improved growth characteristic” as compared to a reference cell or cell culture will have a doubling time which is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater than the doubling time of the reference cell or cell culture when the cells are cultured under the same conditions and for the same period of time.

[0146] In some embodiments, a cell or cell culture that has an “improved growth characteristic” as compared to a reference cell or cell culture will have TXNIP activity which is at least about 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% greater than TXNIP activity of the reference cell or cell culture, when the cells are cultured under the same conditions and for the same period of time.

[0147] In some embodiments, when a cell comprising an exogenous TXNIP gene is cultured, the cell culture exhibits a metabolic shift that is not detected in an otherwise identical cell culture that comprises cells that do not comprise an exogenous TXNIP gene.

[0148] In some embodiments, when a cell that overexpresses a TXNIP gene is cultured,the cell culture exhibits a metabolic shift that is not detected in an otherwise identical cell culture that comprises cells that do not overexpress a TXNIP gene.

[0149] In some embodiments, when a cell that overexpresses a TXNIP polypeptide is cultured, the cell culture exhibits a metabolic shift that is not detected in an otherwise identical cell culture that comprises cells that do not overexpress a TXNIP polypeptide.

[0150] In some embodiments, when a cell that has increased TXNIP activity is cultured, the cell culture exhibits a metabolic shift that is not detected in an otherwise identical cell culture that comprises cells that do not have increased TXNIP activity..

[0151] In some embodiments, when a cell 1) comprising an exogenous TXNIP gene, 2) overexpresses a TXNIP gene, 3) overexpresses a TXNIP polypeptide, 4) has increased TXNIP activity or 5) a combination thereof is cultured, the cell culture exhibits a metabolic shift that is not detected in an otherwise identical culture that comprises cells that do not 1) comprise an exogenous TXNIP gene, 2) overexpress a TXNIP gene, 3) overexpress a TXNIP polypeptide, 4) have increased TXNIP activity or 5) a combination thereof.

[0152] In some embodiments, a metabolic shift in a cell culture is an increase in lactate consumption by the cells, a decrease in lactate production by the cells or a combination of both. In some embodiments, an increase in lactate consumption, a decrease in lactate production or a combination thereof by cells in culture is detected by measuring lactate concentration in media of a cell culture.

[0153] In some embodiments a metabolic shift begins on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.

[0154] In some embodiments, a metabolic shift continues for 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8, days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days 20 days or more of culture.

[0155] In some embodiments, a cell culture that exhibits a metabolic shift comprises less than 0.01 grams lactate per liter cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L of lactate in the media of the cell culture.

[0156] In some embodiments, a cell comprising an exogenous TXNIP gene has increased lactate consumption as compared to an otherwise identical cell that does not comprise an exogenous TXNIP gene. In some embodiments, a cell that overexpresses a TXNIP gene has increased lactate consumption as compared to an otherwise identical cell that does not overexpress a TXNIP gene. In some embodiments, a cell that overexpresses a TXNIP polypeptide has increased lactate consumption as compared to an otherwise identical cell thatdoes not overexpress a TXNIP polypeptide. In some embodiments, a cell that has increased TXNIP activity has increased lactate consumption as compared to an otherwise identical cell that does not have increased TXNIP activity.

[0157] In some embodiments, a cell that comprises an exogenous TXNIP gene, overexpresses a TXNIP gene, overexpresses a TXNIP polypeptide, has increased TXNIP activity or a combination thereof, consumes at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold more lactate as compared lactate consumption of an otherwise identical cell that does not comprise an exogenous TXNIP gene, does not overexpress a TXNIP gene, does not overexpress a TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.

[0158] In some embodiments, a first cell culture comprising a cell that comprises an exogenous TXNIP gene has a lower concentration of lactate in the media of the first cell culture as compared to a second cell culture comprising an otherwise identical cell that does not comprise an exogenous TXNIP gene.

[0159] In some embodiments, a first cell culture comprising a cell that overexpresses a TXNIP gene has a lower concentration of lactate in the media of the first cell culture as compared to a second cell culture comprising an otherwise identical cell that does not overexpress a TXNIP gene.

[0160] In some embodiments, a first cell culture comprising a cell that overexpresses a TXNIP polypeptide has a lower concentration of lactate in the media of the first cell culture as compared to a second cell culture comprising an otherwise identical cell that does not overexpress a TXNIP polypeptide.

[0161] In some embodiments, a first cell culture comprising a cell with increased TXNIP activity has a lower concentration of lactate in the media of the first cell culture as compared to a second cell culture comprising an otherwise identical cell that does not have increased TXNIP activity.

[0162] In some embodiments, a first cell culture comprising a cell that comprises an exogenous TXNIP gene, overexpresses a TXNIP gene, overexpresses a TXNIP polypeptide, has increased TXNIP activity, or a combination thereof, has at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold lower concentration oflactate in the media of the first cell culture as compared to media of a second cell culture comprising an otherwise identical cell that does not comprise an exogenous TXNIP gene, does not overexpress a TXNIP gene, does not overexpress a TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.Cell Culture Methods

[0163] The terms “culture” and “cell culture” as used herein refer to a cell population and a medium under conditions suitable for survival and / or growth of the cell population. As will be clear to those of ordinary skill in the art, in some embodiments, these terms refer to a combination comprising a cell population and medium in which the cell population is suspended. In some embodiments, these terms refer to a combination comprising a cell population and medium in which the cell population is adherent to a surface. In some embodiments, a cell of a cell culture is a mammalian cell. In some embodiments, a cell of a cell culture is a CHO cell.

[0164] The present disclosure may be used with any cell culture method that is amenable to the desired process (e.g., production of a recombinant protein (e.g., antibody)). As an example, cells in culture may be grown in a batch or a fed-batch culture, where the culture is terminated after sufficient expression of a recombinant protein (e.g., antibody), after which the expressed protein (e.g., antibody) is harvested. Alternatively, as another example, cells in culture may be grown in batch-refeed, where the culture is not terminated and new nutrients and other components are periodically or continuously added to the culture, during which an expressed recombinant protein (e.g., antibody) is harvested periodically or continuously. Other suitable methods (e.g., spin-tube cultures) are known in the art and can be used to practice the present disclosure.

[0165] In some embodiments, a cell culture suitable for the present disclosure is a fed- batch culture. The term “fed-batch culture” as used herein refers to a method of culturing cells in which additional components are provided to the culture at a time or times subsequent to the beginning of the culture process. Such provided components typically comprise nutritional components to the cell culture which have been depleted during the culturing process. A fed- batch culture is typically stopped at some point and the cells and / or components in the medium are harvested and optionally purified. In some embodiments, a fed-batch culture comprises a base medium supplemented with feed media.

[0166] A cell culture may be grown in any convenient volume chosen by the practitioner. For example, a cell culture may be grown in small scale reaction vessels ranging in volume from a few milliliters to several liters. Alternatively, a cell culture may be grown in large scale commercial Bioreactors ranging in volume from approximately at least 1 liter (L) to 10 L, 50 L, 100 L, 250 L, 500 L, 1000 L, 2000 L, 2500 L, 5000 L, 8000 L, 10,000 L, 12,000 L, 15000 L,20000 L or 25000 L or more, or any volume in between.

[0167] The temperature of a cell culture will be selected based primarily on the range of temperatures at which the cells in the culture remains viable and the range in which a high level of desired product (e.g., a recombinant protein, e.g., an antibody) is produced. In general, most mammalian cell cultures grow well and can produce desired products (e.g., recombinant proteins) within a range of about 25°C to 42°C, although methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cell cultures grow well and can produce desired products (e.g., recombinant proteins, e.g., antibodies) within the range of about 35°C to 40°C. In certain embodiments, a cell culture is grown at a temperature of 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C or 45°C at one or more times during the cell culture process. Those of ordinary skill in the art will be able to select appropriate temperature or temperatures in which to grow cells in culture, depending on the particular needs of the culture and the particular production requirements of the practitioner. A cell culture may be grown for any amount of time, depending on the needs of the practitioner and the requirement of the cells in the culture. In some embodiment, a cell culture is grown at 37°C. In some embodiments, a cell culture is grown at 36.5°C.

[0168] In some embodiments, a cell culture may be grown during an initial growth phase (or growth phase) for a greater or lesser amount of time, depending on requirements of the cells in the culture, production of a recombinant protein or both. In some embodiments, cells in a culture are grown for a period of time sufficient to achieve a predefined cell density. In some embodiments, cells in a culture are grown for a period of time sufficient to achieve a cell density that is a given percentage of the maximal cell density that the cells would eventually reach if allowed to grow undisturbed. For example, cells in a culture may be grown for a period of time sufficient to achieve a desired viable cell density of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of maximal cell density. In some embodiments, cells in a culture are grown until a cell density does not increase by more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% per day of culture. In some embodiments, cells in a culture are grown until a cell density does not increase by more than 5% per day of culture time.

[0169] In some embodiment, cells in a culture are allowed to grow for a defined period of time. For example, depending on a starting concentration of cells in a culture, a temperature at which the cells are grown, and an intrinsic growth rate of the cells, the cells may be grown for 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days, for example for 4 to 10 days or from 4 to 20 days. In some cases, cells in a culture may be allowed to grow for a month or more. One of ordinary skill in the art would be able to choose a duration of an initial growth phase depending on protein production requirements and needs of the cellsthemselves.

[0170] A cell culture may be agitated or shaken during an initial culture phase in order to increase oxygenation and dispersion of nutrients to the cells. In accordance with the present disclosure, one of ordinary skill in the art would understand that it can be beneficial to control or regulate certain internal conditions of a bioreactor during an initial growth phase, including but not limited to pH, temperature, oxygenation, etc.

[0171] At the end of an initial growth phase, at least one of the cell culture conditions may be shifted so that a second set of culture conditions is applied. In some embodiments, shift of cell culture conditions is associated with a metabolic shift in the culture. A metabolic shift can be accomplished by, e.g., a change in the temperature, pH, osmolality, expression of a particular gene (e.g., TXNIP), chemical inductant level of the cell culture or a combination thereof.

[0172] In some embodiments, cell culture conditions may be shifted by shifting the temperature of the culture. Shifting temperature is not the only mechanism through which a metabolic shift can be achieved. For example, a metabolic shift can also be achieved by shifting other culture conditions including, but not limited to, pH, osmolality, and sodium butyrate levels.

[0173] The timing of a cell culture shift may be determined by one of ordinary skill in the art based on protein production requirements or needs of the cells.

[0174] When shifting a temperature of a culture, the temperature shift may be gradual. For example, it may take several hours or days to complete the temperature change. Alternatively, a temperature shift may be abrupt. For example, a temperature change may be complete in less than several hours. In some embodiments, a temperature change may even be complete within less than an hour.

[0175] In some embodiments, a metabolic shift may occur without a change to culture conditions, e.g., by overexpressing a gene (e.g., TXNIP), overexpression of a polypeptide (e.g., TXNIP). Overexpression of a TXNIP gene, overexpression of a TXNIP polypeptide, increased TXNIP activity or a combination there of may be accomplished by any number of methods including contacting a cell that comprises a TXNIP gene with a molecule that increases transcription of TXNIP mRNA, with a molecule that increases translation of a TXNIP polypeptide, with a molecule that increases TXNIP activity, or a combination thereof. In come embodiments, a molecule that increases expression of a TXNIP gene, increases expression of a TXNIP polypeptide, increases TXNIP activity, or a combination thereof and induces a metabolic shift is glucosamine.

[0176] In some embodiments, a metabolic shift is an increase in lactate consumption by cells in culture, optionally wherein the lactate concentration in the media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactateconcentration in media from a second cell culture media comprising cells that are otherwise identical but that do not overexpress a TXNIP gene, do not overexpress a TXNIP polypeptide, do not have increased TXNIP activity or a combination thereof.

[0177] In some embodiments, a metabolic shift is a decrease in lactate production by cells in culture, optionally wherein the lactate concentration in the media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactate concentration in media from a second cell culture comprising cells that are otherwise identical but that do not overexpress a TXNIP gene, do not overexpress a TXNIP polypeptide, do not have increased TXNIP activity or a combination thereof.

[0178] In some embodiments, a TXNIP gene in a cell (e.g., a mammalian host cell) is overexpressed and induces a metabolic shift when the cell is contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule (e.g., MYC), and the second molecule is an inhibitor of TXNIP gene expression. In some embodiments, a metabolic shift is an increase in lactate consumption by cells in culture, optionally wherein the lactate concentration in the media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactate concentration in media of a second cell culture comprising cells that are otherwise identical but that are not contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule (e.g., MYC), and the second molecule is an inhibitor of TXNIP gene expression. In some embodiments, a metabolic shift is a decrease in lactate production by cells in culture, optionally wherein the lactate concentration in the media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactate concentration in media of a second cell culture comprising cells that are otherwise identical but that are not contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule (e.g., MYC), and the second molecule is an inhibitor of TXNIP gene expression. In some embodiments, a molecule that inhibits MYC activity is JQ1 or OTX-015.

[0179] In some embodiments, a TXNIP polypeptide in a cell (e.g., a mammalian host cell) is overexpressed and induces a metabolic shift when the cell is contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule (e.g., MYC), and the second molecule is an inhibitor of TXNIP polypeptide expression. In some embodiments, a metabolic shift is an increase in lactate consumption by cells in culture, optionally wherein the lactate concentration in the media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactate concentration in media of a second cell culture comprising cells that are otherwise identical but that are not contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule (e.g., MYC), and the second molecule is an inhibitor of TXNIP polypeptide expression. In some embodiments, a metabolic shift is a decrease in lactate production by cells in culture, optionally wherein the lactateconcentration in the media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactate concentration in media of a second cell culture comprising cells that are otherwise identical but that are not contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule (e.g., MYC), and the second molecule is an inhibitor of TXNIP polypeptide expression. In some embodiments, a molecule that inhibits MYC activity is JQ1 or OTX-015.

[0180] In some embodiments, a TXNIP gene, a TXNIP polypeptide or both in a cell (e.g., a mammalian host cell) is overexpressed and induces a metabolic shift when the cell is contacted with a molecule that inhibits MYC activity (e.g., JQ1 , OTX-015). In some embodiments, a metabolic shift is an increase in lactate consumption by cells in culture, optionally wherein the lactate concentration in the media of the cell culture media is decreased, remains constant or does not increase at a rate comparable to the lactate concentration in media of a second cell culture comprising cells that are otherwise identical but that are not contacted with a molecule that inhibits MYC activity (e.g., JQ1 , OTX-015). In some embodiments, a metabolic shift is a decrease in lactate production by cells in culture, optionally wherein the lactate concentration in the media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactate concentration in media of a second cell culture comprising cells that are otherwise identical but that are not contacted with a molecule that inhibits MYC activity (e.g., JQ1 , OTX-015).

[0181] In some embodiments, a TXNIP gene, a TXNIP polypeptide or both in a cell (e.g., a mammalian host cell) is overexpressed and induces a metabolic shift when the cell is contacted with a first molecule that inhibits MYC activity (e.g., JQ1 , OTX-015) and a second molecule that increases expression of a TXNIP gene, a TXNIP polypeptide or both (e.g., glucosamine). In some embodiments, a metabolic shift is an increase in lactate consumption by cells in culture, optionally wherein the lactate concentration in media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactate concentration in media of a second cell culture comprising cells that are otherwise identical but that are not contacted with a first molecule that inhibits MYC activity (e.g., JQ1 , OTX-015) or a second molecule that increase expression of a TXNIP gene, a TXNIP polypeptide or both (e.g., glucosamine). In some embodiments, a metabolic shift is a decrease in lactate production by cells in culture, optionally wherein the lactate concentration in media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactate concentration of media of a second cell culture comprising cells that are otherwise identical but that are not contacted with a first molecule that inhibits MYC activity (e.g., JQ1 , OTX-015) or a second molecule that increase expression of a TXNIP gene, a TXNIP polypeptide or both (e.g., glucosamine).

[0182] In some embodiments, a cell (e.g., a mammalian host cell, e.g., a CHO cell) comprises an exogenous TXNIP gene and induces a metabolic shift. In some embodiments, acell that comprises an exogenous TXNIP gene overexpresses the TXNIP gene, overexpresses a TXNIP polypeptide, has increased TXNIP activity, or a combination thereof and induces a metabolic shift. In some embodiments, a metabolic shift is an increase in lactate consumption by cells in culture, optionally wherein the lactate concentration in media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactate concentration in media of a second cell culture comprising cells do not comprise an exogenous TXNIP gene. In some embodiments, a metabolic shift is a decrease in lactate production by cells in culture, optionally wherein the lactate concentration in media of the cell culture is decreased, remains constant or does not increase at a rate comparable to the lactate concentration in media of a second cell culture comprising cells that do not comprise an exogenous TXNIP gene.

[0183] In some embodiments, once conditions of a cell culture have been shifted as discussed above, the cell culture is maintained for a subsequent production phase under a second set of culture conditions conducive to the survival and viability of the cells in culture and appropriate for expression of a desired therapeutic molecule (e.g., recombinant protein), optionally at commercially adequate levels.

[0184] As discussed above, a cell culture may be shifted by shifting one or more of a number of culture conditions including, but not limited to, temperature, pH, osmolality, expression of a particular gene (e.g., TXNIP) and sodium butyrate levels. In some embodiments, a temperature of a cell culture is shifted. In some embodiments, during a subsequent production phase, a culture is maintained at a temperature or temperature range that is lower than a temperature or temperature range of an initial growth phase. As discussed above, multiple discrete temperature shifts may be employed to increase cell density or viability or to increase expression of a therapeutic molecule (e.g., a recombinant protein).

[0185] In some embodiments, cells in culture may be maintained in a subsequent production phase until a desired cell density or production titer is reached. In some embodiments, cells in culture are allowed to grow for a defined period of time during a subsequent production phase. For example, depending on the number (or titer) of cells in culture at the start of a subsequent growth phase, a temperature at which the cells are grown, and an intrinsic growth rate of the cells, the cells may be grown for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. In some cases, cells in culture may be allowed to grow for a month or more.

[0186] A cell culture may be agitated or shaken during a subsequent production phase in order to increase oxygenation and dispersion of nutrients to the cells. In accordance with the present disclosure, it may be beneficial to control or regulate certain internal conditions of a bioreactor during a subsequent growth phase, including but not limited to pH, temperature,oxygenation, etc.

[0187] In some embodiments, cells in culture express a therapeutic molecule (e.g., a recombinant protein, e.g., an antibody) and the method under which the cells are cultured comprises a growth phase and a production phase.Methods

[0188] In one aspect, the disclosure provides methods of selecting a mammalian host cell (e.g., a CHO cell) having one or more of a reduced synthesis of growth inhibitor, a reduced synthesis of productivity inhibitor, or an improved growth characteristic.

[0189] Methods for selecting a mammalian host cell having one or more of a reduced synthesis of growth inhibitor, a reduced synthesis of productivity inhibitor or an improved growth characteristic comprise a) assaying a mammalian host cell for at least one of 1) overexpression of a TXNIP gene, 2) increased copy number of a TXNIP gene and 3) increased TXNIP activity; b) selecting the host cell comprising at least one of 1) overexpression of a TXNIP gene, 2) increased copy number of a TXNIP gene and 3) increased TXNIP activity.

[0190] A host cell (e.g., a mammalian cell, e.g., a CHO cell) selected by this method and comprising at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity has one or more of: reduced synthesis of growth inhibitor, reduced synthesis of productivity inhibitor or improved growth characteristic as compared to an otherwise identical cell that does not comprise at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity.

[0191] A host cell (e.g., a mammalian cell, e.g., a CHO cell) selected by this method and comprising at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity has a metabolic shift as compared to an otherwise identical cell that does not comprise at least one of 1) overexpression of the TXNIP gene and 2) increased copy number of the TXNIP gene.

[0192] A host cell (e.g., a mammalian cell, e.g., a CHO cell) selected by this method and comprising at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity has one or more of: increased lactate consumption and increased lactate production as compared to an otherwise identical cell that does not comprise at least one of 1) overexpression of the TXNIP gene and 2) increased copy number of the TXNIP gene.

[0193] A host cell (e.g., a mammalian cell, e.g., a CHO cell) selected by this method and comprising at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity, and further comprising an exogenous gene encoding a therapeutic molecule (e.g., a recombinant protein), has increased production of thetherapeutic molecule as compared to an otherwise identical cell that does not comprise at least one of 1 ) overexpression of the TXNIP gene and 2) increased copy number of the TXNIP gene.

[0194] In some embodiments, a cell with overexpression of a TXNIP gene overexpresses a TXNIP polypeptide. In some embodiments, a cell with overexpression of a TXNIP gene has increased TXNIP activity.

[0195] In some embodiments, a cell with an increased copy number of a TXNIP gene overexpresses a TXNIP polypeptide. In some embodiments, a cell with an increased copy number of a TXNIP gene has increased TXNIP activity.

[0196] In some embodiments, a cell with an increased copy number of a TXNIP gene comprises an exogenous TXNIP gene. In some embodiments, an exogenous TXNIP gene encodes a TXNIP polypeptide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 . In some embodiments, an exogenous TXNIP gene comprises a polynucleotide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 2.

[0197] In some embodiments, an exogenous TXNIP gene is chromosomally-integrated in a host cell chromosome.

[0198] In some embodiments, a host cell chromosome contains a recombination target site for site-specific integration of the exogenous nucleotide sequence encoding TXNIP in a host cell chromosome.

[0199] In some embodiments, a host cell comprising an exogenous TXNIP gene also comprises a gene encoding a therapeutic molecule or portion thereof. In some embodiments, a therapeutic molecule is a recombinant protein, e.g., an antibody, an enzyme, a peptide hormone, a fusion protein, a cytokine or a detectable protein.

[0200] In one aspect, the disclosure provides methods of preparing a mammalian host cell (e.g., a CHO cell) having one or more of reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor or an improved growth characteristic.

[0201] Methods for preparing a mammalian host cell having one or more of reduced synthesis of a growth inhibitor, reduced synthesis of productivity inhibitor or an improved growth characteristic thereof comprise introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP into the mammalian cell.

[0202] A host cell comprising an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP has one or more of: reduced synthesis of growth inhibitor, reduced synthesis of productivity inhibitor or an improved growth characteristic as compared to an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.

[0203] In some embodiments, an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP encodes a TXNIP polypeptide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP comprises a polynucleotide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 2.

[0204] In some embodiments, an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP is chromosomally-integrated in a host cell chromosome. In some embodiments, a host cell chromosome contains a recombination target site for site-specific integration of an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP in a host cell chromosome.

[0205] In one aspect, host cells (e.g., CHO cells) and methods disclosed herein may also be used to produce a recombinant therapeutic molecule or portion thereof by providing a host cell with one or more of reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor or an improved growth characteristic, and culturing the host cell under conditions sufficient to produce the recombinant therapeutic molecule. In some embodiments, a recombinant therapeutic molecule is a recombinant protein. In some embodiments, a recombinant protein is an antibody, an enzyme, a peptide hormone, a fusion protein, a cytokine or a detectable protein.

[0206] In some embodiments, when a host cell with improved growth characteristic is cultured a first cell culture is produced wherein the first cell culture has one or more of a greater cell count, a greater cell viability, a greater viable cell count, a greater cell density, or a greater viable cell density, greater cell metabolism, greater cell size, or a combination thereof as compared to a second cell culture comprising an otherwise identical host cell not having an improved growth characteristic, and wherein the first and second cell cultures are grown under the same conditions and for the same time period.

[0207] In one aspect, methods for preparing a mammalian host cell (e.g., a CHO cell) having one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor or an improved growth characteristic comprise reducing activity of a MYC polypeptide in the cell. A host cell having reduced MYC activity has one or more of: reduced synthesis of growth inhibitor(s), reduced synthesis of productivity inhibitor(s), or improved growth characteristic(s) compared to an otherwise identical cell that does not have reduced MYC activity. In some embodiments, activity of a MYC polypeptide in a host cell is reduced by contacting the cell with a compound that inhibits MYC activity, for example by reducing expression of the MYC polypeptide. In some embodiments, a host cell is contacted with acompound that inhibits MYC activity and glucosamine, optionally thereby increasing TXNIP activity, and further optionally inducing a metabolic shift in the cell. In some embodiments, a metabolic shift in a cell in an increase in lactate consumption. In some embodiments, a metabolic shift is a decrease in lactate consumption.Expression of Proteins

[0208] A cell and / or cell culture (e.g., host cell and / or host cell culture, e.g., CHO cell and / or CHO cell culture) is selected or engineered to produce a desired product such as a therapeutic molecule (e.g., recombinant protein, e.g., an antibody). In some embodiments, a cell or cell culture produces a recombinant protein, for example by introduction of a gene encoding the protein of interest and / or by introduction of genetic control elements that regulate expression of that gene (whether endogenous or introduced).

[0209] Variability within a cell population engineered to express a therapeutic molecule may exist such that some cells exhibit more robust growth, produce more therapeutic molecule or a therapeutic molecule with greater activity (e.g., enzymatic activity). In some embodiments, a cell line or clone is selected for robust growth under particular conditions selected for culture and expansion. In some embodiments, a cell engineered to express a particular protein is chosen for large-scale production based on cell growth, final cell density, percent cell viability, titer of the expressed therapeutic molecule or a combination thereof.

[0210] A therapeutic molecule (e.g., recombinant protein) expressed by a host cell, a host cell culture or both, and optionally according to methods and described herein, include any one or more of enzyme, receptor, antibody, hormone, regulatory factor, antigen, binding agent, fusion protein, cytokine, detectable protein etc. A therapeutic molecule (e.g., a recombinant protein) expressed by a host cell, a host cell culture or both, and optionally according to methods described herein, may be used in pharmaceutical compositions. In some embodiments, a recombinant protein expressed by a host cell, host cell culture or both is selected from the group consisting of an antibody, or fragment thereof, nanobody, single domain antibody, glycoprotein, growth factor, clotting factor, cytokine, fusion protein, pharmaceutical drug substance, vaccine and enzyme.Antibodies

[0211] Antibodies and fragments thereof may be expressed in a cell (e.g., a host cell, e.g., a CHO cell) as disclosed herein and which cell may be selected, prepared, produced in accordance with methods as disclosed herein. In some embodiments, an expressed antibody is a monoclonal antibody.

[0212] In some embodiments, a monoclonal antibody is a chimeric antibody. A chimeric antibody contains amino acid fragments that are derived from more than one organism. Chimeric antibody molecules can include, for example, an antigen binding domain from anantibody of a mouse, rat, or other species, with human constant regions. A variety of approaches for making chimeric antibodies have been described. See e.g., Morrison etal., Proc. Natl. Acad. Sci. U.S.A. 81 , 6851 (1985); Takeda etal., Nature 314, 452 (1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi etal., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom Patent GB 2177096B.

[0213] In some embodiments, a monoclonal antibody is a human antibody derived, e.g., through the use of ribosome-display or phage-display libraries (see, e.g., Winter et al., U.S. Patent No. 6,291 ,159 and Kawasaki, U.S. Patent No. 5,658,754). In some embodiments, a monoclonal antibody is a human antibody derived, e.g., through the use of xenographic species in which a native antibody gene is inactivated and functionally replaced with a human antibody gene, while leaving intact other components of the native immune system (see, e.g., Kucherlapati et al., U.S. Patent No. 6,657,103).

[0214] In some embodiments, a monoclonal antibody is a humanized antibody. A humanized antibody is a chimeric antibody wherein a large majority of the amino acid residues are derived from human antibodies, thus minimizing a potential immune reaction when delivered to a human subject. In humanized antibodies, amino acid residues in the complementarity determining regions are replaced, at least in part, with residues from a nonhuman species that confer a desired antigen specificity or affinity. Such altered immunoglobulin molecules can be made by any of several techniques known in the art, e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and may be made according to the teachings of PCT Publication WO92 / 06193 or EP 0239400, all of which are incorporated herein by reference). Humanized antibodies can be commercially produced by, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain. For further reference, see Jones et al., Nature 321 :522-525 (1986); Riechmann etal., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), all of which are incorporated herein by reference.

[0215] In some embodiments, a monoclonal, chimeric, or humanized antibody may contain amino acid residues that do not naturally occur in any antibody in any species in nature. These foreign residues can be utilized, for example, to confer novel or modified specificity, affinity or effector function on the antibody. In some embodiments, an antibody may be conjugated to a drug for systemic pharmacotherapy, such as a toxin, a low-molecular-weight cytotoxic drug, a biological response modifier, and a radionuclide (see e.g., Kunz et aL, Calicheamicin derivative- carrier conjugates, US20040082764 A1).Introduction of Genes for the Expression of Proteins into Host Cells

[0216] Generally, a nucleic acid molecule introduced into a cell encodes a therapeuticmolecule (e.g., a recombinant protein) this is expressed by the cell and later harvested and purified. In some embodiments, a nucleic acid molecule introduced into a cell encodes a gene product that induces expression of a desired protein (e.g., a recombinant protein) by the cell. For example, a nucleic acid molecule introduced into a cell may encode a transcription factor that activates transcription of an endogenous or heterologous protein. In some embodiments, a nucleic acid molecule introduced into a cell may increase translation or stability of a protein expressed by the cell.

[0217] Methods suitable for introducing a nucleic acid into a host cell (e.g., mammalian host cell, e.g., CHO cell) sufficient to achieve expression of a protein encoded by the nucleic acid are known in the art. See, for example, Gething et al., Nature, 293:620-625, 1981 ; Mantei et al., Nature, 281 :40-46, 1979; Levinson et al. EP 117,060; and EP 117,058, each of which is incorporated herein by reference. For a mammalian cell, methods of introducing genetic material into a mammalian cell include the calcium phosphate precipitation method of Graham and van der Erb (Virology, 52:456-457, 1978) or the lipofectamine™ (Gibco BRL) Method of Hawley-Nelson (Focus 15:73, 1993). General aspects of mammalian cell host system transformations have been described by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983. For various techniques for introducing genetic material into mammalian cells, see Keown etal., Methods in Enzymology, 1989, Keown etal., Methods in Enzymology, 185:527-537, 1990, and Mansour etal., Nature, 336:348-352, 1988. Additional methods suitable for introducing a nucleic acid into a cell include electroporation, for example as employed using the GenePulser XCell™ electroporator by BioRad™

[0218] In some embodiments, a nucleic acid introduced into a cell is in the form of a naked nucleic acid molecule. For example, a nucleic acid molecule introduced into a cell may consist only of the nucleic acid encoding a therapeutic molecule (e.g., protein, e.g., recombinant protein) and necessary genetic control elements. Alternatively, a nucleic acid encoding a therapeutic molecule (e.g., protein, e.g., recombinant protein) (including the necessary regulatory elements) may be contained within a plasmid vector. Non-limiting representative examples of suitable vectors for expression of proteins in mammalian cells include pCDNAI ; pCD, see Okayama, et al. Mol. Cell Biol. 5:1136-1142, 1985; pMCIneo Poly-A, see Thomas, et al. Cell 51 :503-512, 1987; a baculovirus vector such as pAC 373 or pAC 610; CDM8 , see Seed, B. Nature 329:840, 1987; and pMT2PC, see Kaufman, et al. EMBO J. 6:187-195, 1987, each of which is incorporated herein by reference in its entirety. In some embodiments, a nucleic acid molecule introduced into a cell is contained within a viral vector. For example, a nucleic acid encoding a therapeutic molecule (e.g., protein, e.g., recombinant protein) may be inserted into a viral genome (or a partial viral genome). Regulatory elements directing expression of a therapeutic molecule (e.g., protein, e.g., recombinant protein) may be included with the nucleic acid inserted into a viral genome (i.e., linked to a gene inserted into a viralgenome) or can be provided by the viral genome itself.

[0219] Naked DNA can be introduced into a cell by forming a precipitate containing the DNA and calcium phosphate. Alternatively, naked DNA can also be introduced into a cell by forming a mixture of the DNA and DEAE-dextran and incubating the mixture with the cell or by incubating the cell and the DNA together in an appropriate buffer, and subjecting the cell to a high-voltage electric pulse (e.g., by electroporation). A further method for introducing naked DNA into a cell is by mixing the DNA with a liposome suspension containing cationic lipids. The DNA / liposome complex is then incubated with the cell. Naked DNA can also be directly injected into a cell by, for example, microinjection. Naked DNA can also be introduced into a cell by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cellsurface receptor (see for example Wu, G. and Wu, C.H. J. Biol. Chem. 263:14621 , 1988;Wilson etal. J. Biol. Chem. 267:963-967, 1992; and U.S. Patent No. 5,166,320, each of which is hereby incorporated by reference in its entirety). Binding of a DNA-ligand complex to a receptor facilitates uptake of the DNA by receptor- mediated endocytosis.

[0220] Use of viral vectors containing particular nucleic acid sequences, e.g., a cDNA encoding a therapeutic molecule (e.g., protein, e.g., recombinant protein), is a common approach for introducing nucleic acid sequences into a cell. Infection of a cell culture with a viral vector comprising a nucleic acid that encodes a therapeutic molecule has the advantage that a large proportion of the cells in the culture receive the nucleic acid. This can obviate the need for selection of cells which have received the nucleic acid. Additionally, molecules encoded within a viral vector, e.g., by a cDNA contained in the viral vector, are generally expressed efficiently in a cell that has taken up viral vector nucleic acid.

[0221] Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. Blood 76:271 , 1990). A recombinant retrovirus can be constructed having a nucleic acid encoding a therapeutic molecule (e.g., protein, e.g., recombinant protein) inserted into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. Such a replication defective retrovirus is then packaged into virions which can be used to infect a target cell through use of a helper virus by standard techniques.

[0222] The genome of an adenovirus can be manipulated such that it encodes and expresses a therapeutic molecule (e.g., protein, e.g., recombinant protein) but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al. BioTechniques 6:616, 1988; Rosenfeld et al. Science 252:431 -434, 1991 ; and Rosenfeld et al. Cell 68:143-155, 1992. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to beeffective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al., 1992, cited supra), endothelial cells (Lemarchand etal., Proc. Natl. Acad. Sci. USA 89:6482-6486, 1992), hepatocytes (Herz and Gerard, Proc. Natl. Acad. Sci. USA 90:2812-2816, 1993) and muscle cells (Quantin et al., Proc. Natl. Acad. Sci. USA 89:2581 -2584, 1992). Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential issues that can occur as a result from insertional mutagenesis (e.g., retroviral DNA). Moreover, the carrying capacity of an adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj- Ahmand and Graham, J. Virol. 57:267, 1986). Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral E1 and E3 genes but retain as much as 80% of the adenoviral genetic material.

[0223] When a method used to introduce a nucleic acid molecule into a population of cells results in modification of a large proportion of the cells, and efficient expression of the therapeutic molecule (e.g., protein, e.g., recombinant protein) by the cells, the modified population of cells may be used without further isolation or subcloning of individual cells within the population. That is, there may be sufficient production of a therapeutic molecule (e.g., protein, e.g., recombinant protein) by the population of cells such that no further cell isolation is needed and the population can immediately be used to seed a cell culture for the production of the therapeutic molecule (e.g., protein, e.g., recombinant protein). Alternatively, it may be desirable to isolate and expand a homogenous population of cells from a few cells or a single cell that efficiently produce the therapeutic molecule (e.g., protein, e.g., recombinant protein).

[0224] An alternative to introducing a nucleic acid molecule into a cell that encodes a therapeutic molecule (e.g., protein, e.g., recombinant protein), the introduced nucleic acid may encode another polypeptide or protein that induces or increases the level of expression of a protein produced endogenously by a cell. For example, a cell may be capable of expressing a particular protein but may fail to do so without additional treatment of the cell. Similarly, a cell may express insufficient amounts of a protein for the desired purpose. Thus, an agent that stimulates expression of a protein of interest can be used to induce or increase expression of that protein by a cell. For example, an introduced nucleic acid molecule may encode a transcription factor that activates or upregulates transcription of a protein of interest. Expression of such a transcription factor in turn leads to expression, or more robust expression of the protein of interest.

[0225] In certain embodiments, a nucleic acid that directs expression of a therapeutic molecule (e.g., protein, e.g., recombinant protein) is stably introduced into a host cell (e.g., a CHO cell). In certain embodiments, a nucleic acid that directs expression of a therapeutic molecule (e.g., protein, e.g., recombinant protein) is transiently introduced into a host cell (e.g.,a CHO cell). One of ordinary skill in the art will be able to choose whether to stably or transiently introduce a nucleic acid into the cell based on experimental needs.

[0226] A gene encoding a therapeutic molecule (e.g., protein, e.g., recombinant protein) may optionally be linked to one or more regulatory genetic control elements. In certain embodiments, a genetic control element directs constitutive expression of the protein. In certain embodiments, a genetic control element provides inducible expression of a gene encoding a therapeutic molecule (e.g., protein, e.g., recombinant protein) can be used. Use of an inducible genetic control element (e.g., an inducible promoter) allows for modulation of production of a therapeutic molecule (e.g., protein, e.g., recombinant protein) in a cell. Non-limiting examples of potentially useful inducible genetic control elements for use in eukaryotic cells include hormone- regulated elements (e.g., see Mader, S. and White, J.H., Proc. Natl. Acad. Sci. USA 90:5603- 5607, 1993), synthetic ligand-regulated elements (see, e.g. Spencer, D.M. et a!., Science 262:1019-1024, 1993) and ionizing radiation-regulated elements (e.g., see Manome, Y. et al., Biochemistry 32:10607-10613, 1993; Datta, R. et al., Proc. Natl. Acad. Sci. USA 89:10149- 10153, 1992). Additional cell-specific or other regulatory systems known in the art may be used in accordance with the disclosure.Isolation of the Expressed Protein

[0227] Therapeutic molecules (e.g., protein, e.g., recombinant protein) expressed by a cell or method of the present disclosure may be isolated, purified or both. In some embodiments, an expressed protein is secreted into cell culture medium by cultured cells. In some embodiments, an expressed protein is retained with a cell of the cell culture. A first step in the purification process may include lysing cells that express a protein and separating the expressed protein from cell and cell fragments by centrifugation, filtration or both. Lysis of mammalian host cells can be achieved by any number of means well known to those of ordinary skill in the art, including physical disruption by glass beads and exposure to high pH conditions.

[0228] An expressed protein may be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation and / or by any other available technique for the purification of proteins (See, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S.J. and Hames, B.D. (eds.), Protein Expression : A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M.P., Simon, M.L, Abelson, J.N. (eds.), Guide to Protein Purification : Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997, each of which is incorporated herein by reference). For immunoaffinity chromatography in particular, a protein may be isolated by binding it to an affinity column comprising antibodies that were raised against that protein and were affixed to a stationary support. Alternatively, affinity tags such as an influenza coat sequence, poly-histidine, orglutathione-S-transferase can be attached to the protein by standard recombinant techniques to allow for easy purification by passage over the appropriate affinity column. Protease inhibitors such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin may be added at any or all stages in order to reduce or eliminate degradation of the protein during the purification process. Protease inhibitors are particularly advantageous when cells must be lysed in order to isolate and purify the expressed protein.Pharmaceutical Compositions

[0229] In certain embodiments, a therapeutic molecule (e.g., recombinant protein, e.g., antibody, cytokine) produced by host cells or methods of the present disclosure will have pharmacologic activity and will be useful in the preparation of a pharmaceutical composition. Pharmaceutical compositions be administered to a subject or may first be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, ophthalmic, transdermal (topical), transmucosal, rectal, and vaginal routes.

[0230] Pharmaceutical compositions include a therapeutic molecule (e.g., recombinant protein, e.g., antibody, cytokine) expressed from a mammalian cell line (e.g., a CHO cell line), a pharmaceutically acceptable carrier, and optionally, a delivery agent (e.g., a cationic polymer, peptide molecular transporter, surfactant, etc.). As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the compositions.

[0231] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0232] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany,NJ) or phosphate buffered saline (PBS). Pharmaceutical compositions should be sterile and fluid to the extent that functional syringability exists. Pharmaceutical compositions are stable under conditions of manufacture and storage and must be preserved against contaminating action of microorganisms such as bacteria and fungi. In general, a relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity of a pharmaceutical composition can be maintained, for example, by the use of a coating such as lecithin, by maintenance of required particle size, in the case of dispersion, and by use of a surfactant. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in a composition. Prolonged absorption of an injectable composition can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and / or gelatin.

[0233] Sterile injectable solutions can be prepared by incorporating a purified therapeutic molecule (e.g., recombinant protein, e.g., antibody, cytokine) in a required amount and in an appropriate solvent with one, or a combination of ingredients enumerated above, followed by filtered sterilization.

[0234] Generally, dispersions are prepared by incorporating a purified therapeutic molecule (e.g., recombinant protein, e.g., antibody, cytokine) expressed from a mammalian cell line into a sterile vehicle which contains a basic dispersion medium and optionally other ingredients such as those described above. Preparation of sterile powders for preparation of sterile injectable solutions may be vacuum dried and / or freeze-dried to yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-f iltered solution thereof.

[0235] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, a purified therapeutic molecule (e.g., recombinant protein, e.g., antibody, cytokine) can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of a pharmaceutical composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Compositions for oral delivery mayadvantageously incorporate agents to improve stability within the gastrointestinal tract and / or to enhance absorption.

[0236] For administration by inhalation, a pharmaceutical composition comprising a purified therapeutic molecule (e.g., recombinant protein, e.g., antibody, cytokine) expressed from a mammalian cell line disclosed herein, or prepared by methods disclosed herein, and a delivery agent are preferably delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. The present disclosure contemplates delivery of a pharmaceutical composition using a nasal spray, inhaler, or other direct delivery to the upper and / or lower airway. According to some embodiments, pharmaceutical compositions comprising a purified therapeutic molecule (e.g., recombinant protein, e.g., antibody, cytokine) expressed from a mammalian cell line (e.g., a CHO cell line) disclosed herein, or prepared by methods disclosed herein, and a delivery agent are formulated as large porous particles for aerosol administration.

[0237] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration of a pharmaceutical composition, penetrants appropriate to the barrier to be permeated are used in the composition. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through use of nasal sprays or suppositories. For transdermal administration, a purified therapeutic molecule (e.g., recombinant protein, e.g., antibody, cytokine) and delivery agents are formulated into ointments, salves, gels, or creams as generally known in the art.

[0238] A pharmaceutical composition can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0239] In some embodiments, a pharmaceutical composition is prepared with carriers that will protect a therapeutic molecule against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0240] It is advantageous to formulate oral or parenteral pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to betreated; each unit containing a predetermined quantity of active therapeutic molecule calculated to produce a desired therapeutic effect in association with a required pharmaceutical carrier.

[0241] A therapeutic molecule (e.g., recombinant protein, e.g., antibody, cytokine) The polypeptide or protein expressed according to the present invention can be administered at various intervals and over different periods of time as required, e.g., one time per week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or 6 weeks, etc. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to severity of a disease or disorder, previous treatments, general health and / or age of a subject, and other diseases present. Generally, treatment of a subject with a therapeutic molecule (e.g., recombinant protein, e.g., antibody, cytokine) as described herein can include a single treatment or, in many cases, can include a series of treatments. It is furthermore understood that appropriate doses may depend upon the potency of a therapeutic molecule and may optionally be tailored to a particular recipient, for example, through administration of increasing doses until a preselected desired response is achieved. It is understood that a specific dose level for any particular subject may depend upon a variety of factors including activity of the therapeutic molecule employed, age, body weight, general health, gender, and diet of a subject, time of administration, route of administration, rate of excretion, any drug combination, and degree of expression or activity to be modulated.

[0242] The present invention includes use of compositions for treatment of nonhuman animals. Accordingly, doses and methods of administration may be selected in accordance with known principles of veterinary pharmacology and medicine. Guidance may be found, for example, in Adams, R. (ed.), Veterinary Pharmacology and Therapeutics, 8thedition, Iowa State University Press; ISBN: 0813817439; 2001.

[0243] Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration (e.g., packaged as a kit).EMBODIMENTS

[0244] Those skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following embodiments (E).E1. A mammalian host cell comprising an exogenous thioredoxin interacting protein (TXNIP) gene.E2. The cell of E1 , wherein the exogenous TXNIP gene encodes a polypeptide comprising an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least97%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:1 .E3. The cell of E1 or E2, wherein the exogenous TXNIP gene is encoded by a nucleic acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:2.E4. The cell of any one of E1 -E3, wherein the TXNIP gene is a mouse TXNIP gene.E5. The cell of any one of E1 -E4, wherein the cell overexpresses the TXNIP gene.E6. The cell of E5, wherein overexpression of the TXNIP gene is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of a TXNIP gene by an otherwise identical cell that does not comprise an exogenous TXNIP gene.E7. The cell of any one of E1 -E6, wherein the cell overexpresses a TXNIP polypeptide.E8. The cell of E7, wherein overexpression of the TXNIP polypeptide is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of a TXNIP polypeptide by an otherwise identical cell that does not comprise an exogenous TXNIP gene.E9. The cell of any one of E1 -E8, wherein the cell has increased TXNIP activity.E10. The cell of E9, wherein the increased TXNIP activity is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than TXNIP activity of an otherwise identical cell that does not comprise an exogenous TXNIP gene.E11. The cell of any one of E1 -E10, wherein the cell further comprises an exogenous gene encoding a therapeutic molecule or portion thereof.E12. The cell of E11 , wherein the therapeutic molecule is a therapeutic polypeptide.E13. The cell of E11 or E12, wherein the portion of the therapeutic molecule is a portion of a therapeutic polypeptide.E14. The cell of any one of E11 -E13, wherein the therapeutic molecule is a recombinant protein.E15. The cell of any one of E11 -E14, wherein the therapeutic molecule is selected from the group consisting of an antibody or fragment thereof (including a single domain antibody),nanobody, glycoprotein, growth factor, clotting factor, cytokine, fusion protein, pharmaceutical drug substance, vaccine, enzyme, receptor, hormone, regulatory factor, antigen, binding agent and detectable protein.E16. The cell of any one of E11 -E15, wherein the cell overexpresses the therapeutic molecule or portion thereof.E17. The cell of E16, wherein overexpression of the therapeutic molecule is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of the therapeutic molecule by an otherwise identical cell that does not comprise an exogenous TXNIP gene.E18. The cell of any one of E15-E17, wherein the therapeutic molecule is an antibody, optionally an IgG antibody.E19. The cell of any one of E15-E18, wherein when the cell is cultured in a cell culture, the cell produces at least 0.01 g antibody per L cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1 .2 g / L, 1 .4 g / L, 1 .6 g / L, 1 .8 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L. 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 / g / L, 20 g / L or more.E20. The cell of any one of E1 -E19, wherein the cell is a mouse cell, a rat cell, a Chinese Hamster Ovary (CHO) cell or a human cell.E21. The cell of any one of E1 -E20, wherein the cell is a CHO cell comprising an exogenous thioredoxin interacting protein (TXNIP) gene and an exogenous gene encoding a therapeutic molecule or portion thereof.E22. A mammalian host cell, wherein the host cell overexpresses a TXNIP gene, overexpresses a TXNIP polypeptide, has increased TXNIP activity or a combination thereof. E23. The cell of E22, wherein the cell comprises an exogenous TXNIP gene.E24. The cell of E22 or E23, wherein overexpression of the TXNIP gene, overexpression of the TXNIP polypeptide, increased TXNIP activity or a combination thereof is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of a TXNIP gene, expression of a TXNIP polypeptide, TXNIP activity or a combination thereof by an otherwise identical cell.E25. The cell of any one of E22-E24, wherein the cell further comprises an exogenous gene encoding a therapeutic molecule or portion thereof.E26. The cell of E25, wherein the therapeutic molecule is a therapeutic polypeptide.E27. The cell of E25 or E26, wherein the portion of the therapeutic molecule is a portion of a therapeutic polypeptide.E28. The cell of any one of E25-E27, wherein the therapeutic molecule is a recombinant protein.E29. The cell of any one of E25-E28, wherein the therapeutic molecule is selected from the group consisting of an antibody or fragment thereof (including a single domain antibody), nanobody, glycoprotein, growth factor, clotting factor, cytokine, fusion protein, pharmaceutical drug substance, vaccine, enzyme, receptor, hormone, regulatory factor, antigen, binding agent and detectable protein.E30. The cell of any one of E25-E29, wherein the cell overexpresses the therapeutic molecule or portion thereof.E31. The cell of E30, wherein overexpression of the therapeutic molecule is at least 1.5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of the therapeutic molecule by an otherwise identical cell that does not overexpress a TXNIP gene, does not overexpress a TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.E32. The cell of any one of E25-E31 , wherein the therapeutic molecule is an antibody, optionally an IgG antibody.E33. The cell of E32, wherein when the cell is cultured in a cell culture, the cell produces at least 0.01 g antibody per L cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1 .2 g / L, 1 .4 g / L, 1 .6 g / L, 1 .8 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L. 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L, 20 g / L or more when cultured in a cell culture.E34. The cell of any one of E22-E33, wherein the cell is a mouse cell, a rat cell, a Chinese Hamster Ovary (CHO) cell or a human cell.E35. The cell of any one of E22-E34, wherein the cell is a CHO cell comprising an exogenous gene encoding a therapeutic molecule or portion thereof.E36. The cell of E1-E35, wherein the TXNIP gene in the cell is overexpressed when the cell is contacted with a molecule that increases transcription of TXNIP mRNA as compared to TXNIP gene expression by an otherwise identical cell that is not contacted with the molecule that increases transcription of TXNIP mRNA.E37. The cell of E36, wherein the TXNIP gene in the cell is overexpressed by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold,at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater as compared to expression of a TXNIP gene by an otherwise identical cell that is not contacted with the molecule that increases transcription of TXNIP mRNA.E38. The cell of E36 or E37, wherein the molecule that increases transcription of TXNIP mRNA is glucosamine.E39. The cell of E1-E38, wherein a TXNIP polypeptide is overexpressed when the cell is contacted with a molecule that increases translation of the TXNIP polypeptide as compared to TXNIP polypeptide expression by an otherwise identical cell that is not contacted with the molecule that increases translation of the TXNIP polypeptide.E40. The cell of E39, wherein the TXNIP polypeptide in the cell is overexpressed by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold as compared to expression of a TXNIP polypeptide by an otherwise identical cell that is not contacted with the molecule that increases translation of the TXNIP polypeptide.E41. The cell of E39 or E40, wherein the molecule that increases translation of a TXNIP polypeptide is glucosamine.E42. The cell of any one of E1 -E41 , wherein the cell is a CHO cell.E43. The cell of any one of E1 -E21 , E23-E42, wherein the exogenous TXNIP gene in the cell is overexpressed when the cell is contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule, and the second molecule is an inhibitor of TXNIP gene expression.E44. The cell of E43, wherein the TXNIP gene in the cell is overexpressed by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to expression of a TXNIP gene by an otherwise identical cell that is not contacted with the first molecule that inhibits, reduces or decreases activity of the second molecule, and the second molecule is an inhibitor of TXNIP gene expression.E45. The cell of E43 or E44, wherein the first molecule that inhibits, reduces or decreases activity of a second molecule is JQ1 , OTX-015, MYCM1 -6, EN4, KJPyr9, APTO-253, 10058-F4, Jy-3-094, MYCi361 or a combination thereof.E46. The cell of any one of E43-E45, wherein the second molecule is a MYC polypeptide.E47. The cell of any one of E1 -E46, wherein a TXNIP polypeptide is overexpressed when the cell is contacted with a first molecule that inhibits, reduces or decreases activity of a second molecule, and the second molecule is an inhibitor of TXNIP polypeptide expression.E48. The cell of E47, wherein the TXNIP polypeptide in the cell is overexpressed by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold as compared to expression of a TXNIP polypeptide by an otherwise identical cell that is not contacted with the first molecule that inhibits, reduces or decreases activity of the second molecule, and the second molecule is an inhibitor of TXNIP gene expression.E49. The cell of E47 or E48, wherein the first molecule that inhibits, reduces or decreases activity of a second molecule is JQ1 , OTX-015, MYCM1 -6, EN4, KJPyr9, APTO-253, 10058-F4, Jy-3-094, MYCi361 or a combination thereof.E50. The cell of any one of E47-E49, wherein the second molecule is a MYC polypeptide.E51. The cell of any one of E1 -E21 , E23-E50, wherein the exogenous TXNIP gene in the cell is overexpressed when the cell is contacted with a molecule that inhibits MYC activity.E52. The cell of E51 , wherein the exogenous TXNIP gene in the cell is overexpressed by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold as compared to expression of a TXNIP gene by an otherwise identical cell that is not contacted with the molecule that inhibits MYC activity.E53. The cell of E51 or E52, wherein the molecule that inhibits MYC activity is JQ1 , OTX- 015, MYCM1-6, EN4, KJPyr9, APTO-253, 10058-F4, Jy-3-094, MYCi361 or a combination thereof.E54. The cell of any one of E1-E53, wherein a TXNIP polypeptide is overexpressed when the cell is contacted with a molecule that inhibits MYC activity.E55. The cell of E54, wherein the TXNIP polypeptide in the cell is overexpressed by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold as compared to expression of a TXNIP polypeptide by an otherwise identical cell that is not contacted with the molecule that inhibits MYC activity.E56. The cell of E54 or E55, wherein the molecule that inhibits MYC activity is JQ1 , OTX-015, MYCM1-6, EN4, KJPyr9, APTO-253, 10058-F4, Jy-3-094, MYCi361 or a combination thereof.E57. The cell of any one of E1 -E21 ,E23-E56, wherein the exogenous TXNIP gene in the cell is overexpressed when the cell is contacted with a first molecule that inhibits MYC activity and a second molecule that increases expression of the TXNIP gene.E58. The cell of E57, wherein the TXNIP gene in the cell is overexpressed by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to expression of a TXNIP gene by an otherwise identical cell that is not contacted with the first molecule that inhibits MYC activity and the second molecule that increases expression of the TXNIP gene.E59. The cell of E57 or E58, wherein the molecule that inhibits MYC activity is JQ1 , OTX- 015, MYCM1-6, EN4, KJPyr9, APTO-253, 10058-F4, Jy-3-094, MYCi361 or a combination thereof.E60. The cell of any one of E57-E59, wherein the molecule that increases expression of the TXNIP gene is glucosamine.E61. The cell of any one of E1 -E61 , wherein a TXNIP polypeptide is overexpressed when the cell is contacted with a first molecule that inhibits MYC activity and a second molecule that increases expression of the TXNIP polypeptide.E62. The cell of E61 , wherein the TXNIP polypeptide in the cell is overexpressed by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold as compared to expression of a TXNIP polypeptide by an otherwise identical cell that is not contacted with the first molecule that inhibits MYC activity and the second molecule that increases expression of the TXNIP polypeptide.E63. The cell E61 or E62, wherein the molecule that inhibits MYC activity is JQ1 , OTX-015, MYCM1 -6, EN4, KJPyr9, APTO-253, 10058-F4, Jy-3-094, MYCi361 or a combination thereof. E64. The cell of any one of E61 -E63, wherein the molecule that increases expression of the TXNIP polypeptide is glucosamine.E65. The cell of any one of E1 -E64, wherein the cell has one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic.E66. The cell of E65, wherein the synthesis of a growth inhibitor is reduced, the synthesis ofa productivity inhibitor is reduced, or a growth characteristic is improved as compared to synthesis of a growth inhibitor, synthesis or a productivity inhibitor or a growth characteristic by an otherwise identical cell that does not comprise an exogenous TXNIP gene, does not overexpress a TXNIP gene, does not overexpress a TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.E67. The cell of E65 or E66, wherein synthesis of the growth inhibitor is reduced by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold as compared to synthesis of a growth inhibitor by an otherwise identical cell that does not comprise an exogenous TXNIP gene, does not overexpress a TXNIP gene, does not overexpress a TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.E68. The cell of any one of E65-E67, wherein when the cell is cultured to produce a first cell culture, the concentration of the growth inhibitor is measured on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E69. The cell of any one of E65-E68, wherein the grown inhibitor is a cell metabolite.E70. The cell of E69, wherein the cell metabolite results from metabolism of amino acids, carbohydrates, fatty acids or a combination thereof.E71. The cell of E69 or E70, wherein the cell metabolite is selected from the group consisting 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate (indole-3- lactate), indolecarboxylic acid (indole-3-carboxylic acid), homocysteine, 2-hydroxybutyric acid, isovalerate, butyrate, isobutyrate, 2-methylbutyrate, formate, lactate and a combination thereof. E72. The cell of any one of E69-E71 , wherein the cell metabolite is lactate.E73. The cell of E72, wherein media of the first cell culture comprises less than 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L of lactate.E74. The cell of any one of E65-E73, wherein synthesis of the productivity inhibitor is reduced by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to synthesis of a productivity inhibitor by an otherwise identical cell that does not comprise an exogenous TXNIP gene, does not overexpress a TXNIP gene, doesnot overexpress a TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.E75. The cell of any one of E65-E74, wherein when the cell is cultured to produce a first cell culture, the concentration of the productivity inhibitor is measured on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E76. The cell of any one of E65-E75, wherein the productivity inhibitor is a cell metabolite. E77. The cell of E76, wherein the cell metabolite results from metabolism of amino acids, carbohydrates, fatty acids or a combination thereof.E78. The cell of E76 or E77, wherein the cell metabolite is selected from the group consisting of 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate (indole-3- lactate), indolecarboxylic acid (indole-3-carboxylic acid), homocysteine, 2-hydroxybutyric acid, isovalerate, butyrate, isobutyrate, 2-methylbutyrate, formate, lactate and a combination thereof. E79. The cell of any one of E76-E78, wherein the cell metabolite is lactate.E80. The cell of E75, wherein the media of the first cell culture comprises less than 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L,9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L of lactate.E81. The cell of any one of E65-E80, wherein the growth characteristic is improved by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold as compared to a growth characteristic of an otherwise identical cell that does not comprise an exogenous TXNIP gene, does not overexpress a TXNIP gene, does not overexpress a TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.E82. The cell of E65-E81 , wherein when the cell is cultured to produce a first cell culture, the growth characteristic is measured on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E83. The cell of any one of E65-E82, wherein the growth characteristic of the first cell culture that is improved is selected from the group consisting of cell count (also known as “cell number”), cell viability, viable cell count, cell density, viable cell density, cell metabolism, cell size, or a combination thereof.E84. The cell of E82 or E83, wherein the growth characteristic of the first cell culture is improved by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%,200%, 300%, or 500% as compared to the growth characteristic of a second cell culture comprising cells that do not comprise an exogenous TXNIP gene, do not overexpress a TXNIP gene, do not overexpress a TXNIP polypeptide, do not have increased TXNIP activity or a combination thereof.E85. The cell of E83 or E84, wherein the cell is a CHO cell comprising an exogenous TXNIP gene, and when cultured produces a first cell culture that has an increased viable cell density as compared to a viable cell density of a second cell culture comprising an otherwise identical cell that does not comprise an exogenous TXNIP gene.E86. The cell of E85, wherein the viable cell density of the first cell culture is increased by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to a viable cell density of a second cell culture comprising an otherwise identical cell that does not comprise an exogenous TXNIP gene.E87. The cell of any one of E83-E86, wherein the viable cell density of the first cell culture is at least 5 x 105cells per milliliters cell culture media (cells / mL), 10 x 105cells / mL, 25 x 105cells / mL, 50 x 105cells / mL, 100 x 105cells / mL, 150 x 105cells / mL, 200 x 105cells / mL, 250 x 105cells / mL, 300 x 105cells / mL, 350 x 105cells / mL, 400 x 105cells / mL, 450 x 5005cells / mL, 550 x 105cells / mL, 600 x 105cells / mL or 650 x 105cells / mL.E88. The cell of any one of E1 -E87, wherein the cell exhibits a metabolic shift.E89. The cell of E88, wherein the metabolic shift comprises increased lactate consumption, decreased lactate production or a combination thereof.E90. The cell of E89, wherein the increased lactate consumption, decreased lactate production or combination thereof is detected by measuring a lactate concentration in the media of a cell culture comprising the cell.E91. The cell of E90, wherein the lactate concentration in the media of a cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold lower than the lactate concentration in media of a second cell culture comprising cells that do not comprise an exogenous TXNIP gene, do not overexpress a TXNIP gene, do not overexpress a TXNIP polypeptide, do not have increased TXNIP activity or a combination thereof.E92. The cell of any one of E88-E91 , wherein when the cell is cultured, the metabolic shift begins on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of cell culture.E93. The cell of any one of E88-E92, wherein when the cell is cultured, the metabolic shift continues for 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6days, 7 days, 8, days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days 20 days or more of cell culture.E94. The cell of any one of E90-E93, wherein the lactate concentration in media of the cell culture is less than 0.01 grams lactate per liter of cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L.E95. The cell of any one of E88-E94, wherein the cell is a CHO cell comprising an exogenous TXNIP gene and exhibits a metabolic shift comprising increased lactate consumption, decreased lactate production or a combination thereof as compared to lactate consumption, lactate production of a combination thereof of an otherwise identical cell that does not comprise an exogenous TXNIP gene.E96. The cell of any one of E1 -E95, wherein the cell is a CHO cell comprising an exogenous TXNIP gene and when cultured, produces a cell culture with an increased viable cell density and exhibits a metabolic shift comprising increased lactate consumption, decreased lactate production or a combination thereof as compared to the viable cell density, lactate consumption, lactate production or a combination thereof of a second cell culture comprising an otherwise identical cell that does not comprise an exogenous TXNIP gene.E97. A method of selecting a mammalian host cell having one or more of: reduced synthesis of growth inhibitor, reduced synthesis of productivity inhibitor, or improved growth characteristic comprising a) assaying a mammalian host cell for at least one of 1 ) overexpression of a TXNIP gene, 2) increased copy number of a TXNIP gene and 3) increased TXNIP activity and b) selecting the host cell comprising at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity. E98. The method of E97, the wherein the host cell comprising at least one of 1 ) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity has one or more of reduced synthesis of growth inhibitor, reduced synthesis of productivity inhibitor, or improved growth characteristic as compared to synthesis of a growth inhibitor, synthesis of a productivity inhibitor or a growth characteristic of an otherwise identical cell that does not comprise at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity.E99. The method of E97 or E98, wherein synthesis of the growth inhibitor is reduced by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold as compared to synthesis of a growth inhibitor by an otherwise identical cell that doesnot comprise overexpression of the TXNIP gene, does not have an increased copy number of the TXNIP gene, does not have increased TXNIP activity or a combination thereof.E100. The method of any one of E97-E99, further comprising culturing the host cell to produce a first cell culture.E101. The method of E100, further comprising measuring a concentration of the growth inhibitor in the media of the first cell culture on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E102. The method of any one of E97-E101 , wherein the grown inhibitor is a cell metabolite. E103. The method of E102, wherein the cell metabolite results from metabolism of amino acids, carbohydrates, fatty acids or a combination thereof.E104. The method of E102 or E103, wherein the cell metabolite is selected from the group consisting 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate (indole-3-lactate), indolecarboxylic acid (indole-3-carboxylic acid), homocysteine, 2- hydroxybutyric acid, isovalerate, butyrate, isobutyrate, 2-methylbutyrate, formate, lactate, ammonia and a combination thereof.E105. The method of any one of E102-E104, wherein the cell metabolite is lactate.E106. The method of E105, wherein less than 0.01 grams per liter cell media (g / L), 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L of lactate is present in the media of the first cell culture.E107. The method of any one of E97-E106, wherein synthesis of the productivity inhibitor is reduced by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to synthesis of a productivity inhibitor by an otherwise identical cell that does not comprise overexpression of the TXNIP gene, does not have increased copy number of the TXNIP gene, does not have increased TXNIP activity or a combination thereof.E108. The method of any one of E100-E107, further comprising measuring the concentration of the productivity inhibitor in the media of the first cell culture on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E109. The method of any one of E97-E108, wherein the productivity inhibitor is a cell metabolite.E110. The method of E109, wherein the cell metabolite results from metabolism of aminoacids, carbohydrates, fatty acids or a combination thereof.E111. The method of E109 or E110, wherein the cell metabolite is selected from the group consisting 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate (indole-3-lactate), indolecarboxylic acid (indole-3-carboxylic acid), homocysteine, 2- hydroxybutyric acid, isovalerate, butyrate, isobutyrate, 2-methylbutyrate, formate, lactate, ammonia and a combination thereof.E112. The method of any one of E109-E111 , wherein the cell metabolite is lactate.E113. The method of E112, wherein less than 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L of lactate is present in the media of the first cell culture.E114. The method of any one of E97-E113, wherein the growth characteristic is improved by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to a growth characteristic of an otherwise identical cell that does not comprise overexpression of the TXNIP gene, does not have increased copy number of the TXNIP gene, does not have increased TXNIP activity or a combination thereof.E115. The method of any one of E100-E115, further comprising measuring the growth characteristic of the cell or first cell culture on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E116. The method of any one of E100-E115, wherein the growth characteristic of the cell or the first cell culture that is improved is selected from the group consisting of cell count (also known as “cell number”), cell viability, viable cell count, cell density, viable cell density, cell metabolism, cell size, and a combination thereof.E117. The method of E97-E116, wherein the growth characteristic of the cell or first cell culture is improved by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to the growth characteristic of a second cell or second cell culture comprising an otherwise identical cell that does not comprise overexpression of the TXNIP gene, does not have increased copy number of the TXNIP gene, does not have increased TXNIP activity or a combination thereof.E118. The method of E116-E117, wherein the host cell is a CHO cell comprising an increased copy number of the TXNIP gene, and when cultured, produces a first cell culture that has an increased viable cell density as compared to a viable cell density of a second cell culture comprising an otherwise identical cell that does not comprise an increased copy number of theTXNIP gene.E119. The method of E1 18, wherein the viable cell density of the first cell culture is increased by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300% or 500% as compared to a viable cell density of a second cell culture comprising an otherwise identical cell that does not comprise an increased copy number of the TXNIP gene.E120. The method of any one of E1 16-E119, wherein the viable cell density is at least 5 x 105cells per mL cell culture media (cells / mL), 10 x 105cells / mL, 25 x 105cells / mL, 50 x 105cells / mL, 100 x 105cells / mL, 150 x 105cells / mL, 200 x 105cells / mL, 250 x 105cells / mL, 300 x 105cells / mL, 350 x 105cells / mL, 400 x 105cells / mL, 450 x 5005cells / mL, 550 x 105cells / mL, 600 x 105cells / mL or 650 x 105cells / mL.E121. The method of any one of E97-E120, wherein the exogenous TXNIP gene encodes a polypeptide comprising an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:1.E122. The method of any one of E97-E121 , wherein the exogenous TXNIP gene is encoded by a nucleic acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:2.E123. The method of any one of E97-E122, further comprising introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a therapeutic molecule into the host cell.E124. A method of preparing a mammalian host cell having one or more of reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic, the method comprising: introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP into the mammalian cell, wherein the host cell comprising the exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP has one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic compared to an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E125. The method of E124, wherein the exogenous nucleotide sequence encoding TXNIP is chromosomally-integrated in a host cell chromosome.E126. The method of E124 or E125, wherein the host cell chromosome contains a recombination target site for site-specific integration of the exogenous nucleotide sequence encoding TXNIP in a host cell chromosome.E127. The method of any one of E124-E126, further comprising introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a therapeutic molecule intothe host cell.E128. The method of E127, wherein the exogenous nucleotide sequence encoding a therapeutic molecule is chromosomally-integrated in a host cell chromosome.E129. The method of E128, wherein the host cell chromosome contains a recombination target site for site-specific integration of the exogenous nucleotide sequence encoding the therapeutic molecule in a host cell chromosome.E130. The method of any one of E127-E129, wherein the therapeutic molecule is an antibody (e.g., IgG antibody).E131. The method of any one of E124-E130, further comprising culturing the host cell to produce a first cell culture.E132. The method of E131 , wherein the first cell culture produces an increased amount of therapeutic molecule as compared to the amount of therapeutic molecule produced by a second cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E133. The method of E132, wherein the amount of therapeutic molecule produced by the first cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than the amount of therapeutic molecule produced by a second cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E134. The method of E132 or E133, wherein the amount of therapeutic molecule (e.g. antibody) produced by the first cell culture is at least 0.01 g antibody per L cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1 .2 g / L, 1 .4 g / L, 1 .6 g / L, 1 .8 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L. 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L, 20 g / L or more.E135. The method of any one of E124-E134, wherein synthesis of the growth inhibitor is reduced by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to synthesis of a growth inhibitor by an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E136. The method of any one of E131 -E135, further comprising measuring the concentration of the growth inhibitor in the media of the first cell culture on day 1 , day 2, day 3, day 4, day 5,day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E137. The method of any one of E124-E136, wherein the grown inhibitor is a cell metabolite. E138. The method of E137, wherein the cell metabolite results from metabolism of amino acids, carbohydrates, fatty acids or a combination thereof.E139. The method of E137 or E138, wherein the cell metabolite is selected from the group consisting 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate (indole-3-lactate), indolecarboxylic acid (indole-3-carboxylic acid), homocysteine, 2- hydroxybutyric acid, isovalerate, butyrate, isobutyrate, 2-methylbutyrate, formate, lactate, ammonia, and a combination thereof.£140. The method of any one of E137-E139, wherein the cell metabolite is lactate.E141. The method of E140, wherein less than 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L of lactate is present in the media of the first cell culture.E142. The method of any one of E124-E141 , wherein synthesis of the productivity inhibitor is reduced by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to synthesis of a productivity inhibitor by an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E143. The method of any one of E131 -E142, further comprising measuring the concentration of the productivity inhibitor in the media of the first cell culture on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E144. The method of any one of E124-E143, wherein the productivity inhibitor is a cell metabolite.E145. The method of E144, wherein the cell metabolite results from metabolism of amino acids, carbohydrates, fatty acids or a combination thereof.E146. The method of E144 or E145, wherein the cell metabolite is selected from the group consisting 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate (indole-3-lactate), indolecarboxylic acid (indole-3-carboxylic acid), homocysteine, 2- hydroxybutyric acid, isovalerate, butyrate, isobutyrate, 2-methylbutyrate, formate, lactate, ammonia and a combination thereof.E147. The method of any one of E144-E146, wherein the cell metabolite is lactate.E148. The method of E147, wherein less than 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L of lactate is present in media of the first cell culture.E149. The method of any one of E124-E148, wherein the growth characteristic is improved by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to a growth characteristic of an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E150. The method of any one of E131 -E150, further comprising measuring the growth characteristic of the cell or first cell culture on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E151. The method of any one of E124-E150, wherein the growth characteristic of the cell or the first cell culture that is improved is selected from the group consisting of cell count (also known as “cell number”), cell viability, viable cell count, cell density, viable cell density, cell metabolism, cell size, any a combination thereof.E152. The method of E150 or E151 , wherein the growth characteristic of the cell or first cell culture is improved by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to the growth characteristic of an otherwise identical cell or a second cell culture that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E153. The method of E151 or E152, wherein the host cell is a CHO cell and when cultured produces a first cell culture that has an increased viable cell density as compared to a viable cell density of a second cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E154. The method of E153, wherein the viable cell density of the first cell culture is increased by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to the viable cell density of a second cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E155. The method of any one of E151 -E154, wherein the viable cell density of first cell culture is at least 5 x 105cells per milliliter cell culture media (cells / mL), 10 x 105cells / mL, 25 x 105cells / mL, 50 x 105cells / mL, 100 x 105cells / mL, 150 x 105cells / mL, 200 x 105cells / mL, 250 x 105cells / mL, 300 x 105cells / mL, 350 x 105cells / mL, 400 x 105cells / mL, 450 x 5005cells / mL, 550 x 105cells / mL, 600 x 105cells / mL or 650 x 105cells / mLE156. The method of any one of E124-E155, wherein the nucleotide sequence encoding TXNIP encodes a polypeptide comprising an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:1 .E157. The method of any one of E124-E156, wherein the nucleotide sequence encoding TXNIP is encoded by a nucleic acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:2.E158. A method of preparing a mammalian host cell culture having one or more of: 1) an improved growth characteristic and 2) a characteristic of exhibiting a metabolic shift, the method comprising, introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP into a mammalian host cell and culturing the mammalian host cell to produce a first host cell culture, wherein the first host cell culture has one or more of: 1) an improved growth characteristic and 2) exhibits a metabolic shift as compared to a second host cell culture comprising a mammalian host cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E159. The method of E158, wherein the exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP encodes a polypeptide comprising an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:1 .E160. The method of E158 or E159, wherein the exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP is encoded by a nucleic acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:2.E161. The method of any one of E158-E160, wherein the TXNIP nucleotide sequence is a mouse TXNIP nucleotide sequence.E162. The method of any one of E158-E161 , wherein the mammalian host cell overexpresses the TXNIP nucleotide sequence.E163. The method of any one of E158-E162, wherein the exogenous nucleotide molecule encoding TXNIP is chromosomally-integrated in the mammalian host cell chromosome.E164. The method of E163, wherein the host cell chromosome contains a recombination target site for site-specific integration of the exogenous nucleotide sequence encoding TXNIP in themammalian host cell chromosome.E165. The method of any one of E158-E164, wherein the growth characteristic that is improved is selected from the group consisting of cell count (also known as “cell number”), cell viability, viable cell count, cell density, viable cell density, cell metabolism, cell size, and a combination thereof.E166. The method of any one of E158-E164, wherein the growth characteristic of the first host cell culture is improved by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least90 fold, at least 95 fold at least 100 fold as compared to a growth characteristic of the second host cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E167. The method of any one of E158-E166, wherein the growth characteristic of the first host cell culture is improved by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to a growth characteristic of the second host cell culture comprising a cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E168. The method of any one of E158-E167, wherein the growth characteristic is measured on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 1 1 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E169. The method of E165-E168, wherein the first host cell culture has an increased viable cell density as compared to a viable cell density of the second host cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E170. The method of E169, wherein the viable cell density of the first host cell culture is increased by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to a viable cell density of the second host cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E171. The method of any one of E165-E170, wherein the viable cell density of the first cell culture is at least 5 x 105cells / mL, 10 x 105cells / mL, 25 x 105cells / mL, 50 x 105cells / mL, 100 x 105cells / mL, 150 x 105cells / mL, 200 x 105cells / mL, 250 x 105cells / mL, 300 x 105cells / mL, 350 x 105cells / mL, 400 x 105cells / mL, 450 x 5005cells / mL, 550 x 105cells / mL, 600 x 105cells / mL, 650 x 105cells / mL.E172. The method of any one of E158-E171 , wherein the metabolic shift comprises an increased lactate consumption, a decreased lactate production or a combination thereof.E173. The method of E172, wherein the increased lactate consumption, decreased lactate production or combination thereof is detected by measuring a lactate concentration in media of the first host cell culture.E174. The method of E173, wherein the lactate concentration in the first host cell culture media is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold lower than a lactate concentration in the second host cell culture media. E175. The method of any one of E158-E174, wherein the metabolic shift of the first host cell culture begins on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E176. The method of any one of E158-E175, wherein the metabolic shift continues for 6 hours,8 hours, 12 hours, 16 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8, days,9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days 20 days or more of culture.E177. The method of any one of E174-E176, wherein the lactate concentration in the first cell culture media is less than 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L.E178. The method of any one of E158-E177, wherein the first host cell culture exhibits a metabolic shift comprising increased lactate consumption, decreased lactate production or a combination thereof as compared to lactate consumption, lactate production or a combination thereof of a second host cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E179. The method of any one of E158-E178, wherein the first host cell culture has an increased viable cell density and exhibits a metabolic shift comprising increased lactate consumption, decreased lactate production or a combination thereof as compared to a viable cell density, lactate consumption, lactate production or a combination thereof of a second host cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E180. The method of any one of E158-E180, further comprising introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a therapeutic molecule into the host cell.E181. The method of E180, wherein the therapeutic molecule is produced by the host cell, the first host cell culture or both.E182. The method of E181 , wherein the amount of therapeutic molecule produced by the first host cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than the amount of therapeutic molecule produced by the second host cell culture comprising an otherwise identical host cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.E183. The method of any one of E180-E182, wherein the therapeutic molecule is an antibody, optionally an IgG antibody.E184. The method of E183, wherein the first host cell culture produces at least 0.01 g antibody per L cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1 .2 g / L, 1 .4 g / L, 1 .6 g / L, 1 .8 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L. 7 g / L, 8 g / L, 9 g / L 10 g / L, 15 / g / L or 20 g / L.E185. A method for producing a metabolic shift in a host cell, the method comprising, introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP into the host cell, and optionally, wherein the host cell is a CHO cell.E186. The method of E185, further comprising contacting the host cell comprising the exogenous TXNIP gene with a molecule that increases expression of the TXNIP gene in the cell, increases expression of a TXNIP polypeptide in the cell, increases TXNIP activity in the cell or a combination thereof.E187. A method for producing a metabolic shift in a host cell, the method comprising, contacting a host cell comprising a TXNIP gene with a molecule that increases expression of the TXNIP gene in the cell, increases expression of a TXNIP polypeptide in the cell, increases TXNIP activity in the cell or a combination thereof, and optionally, wherein the host cell is a CHO cell.E188. The method of any one of E185-E187, wherein the metabolic shift comprises an increased lactate consumption, a decreased lactate production or a combination thereof as compared to lactate consumption, lactate production or a combination thereof in an otherwise identical host cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP, is not contacted with a molecule that increases expression of a TXNIP gene in the cell, is not contacted with a molecule that increases expression of a TXNIP polypeptide in the cell, is not contacted with a molecule that increases TXNIP activity in the cell or a combination thereof.E189. The method of any one of E185-E188, further comprising culturing the host cell in a host cell culture, and measuring lactate concentration in media of the host cell culture.E190. The method of E189, wherein the lactate concentration in the media of the host cellculture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold lower than a lactate concentration in media of a second host cell culture comprising an otherwise identical host cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP, is not contacted with a molecule that increases expression of a TXNIP gene in the cell, is not contacted with a molecule that increases expression of a TXNIP polypeptide in the cell, is not contacted with a molecule that increases TXNIP activity in the cell or a combination thereof.E191. The method of E189 or E190, wherein the lactate concentration in the host cell culture media is less than 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L.E192. The method of any one of E185-E192, wherein the molecule that increases expression of a TXNIP gene in the cell, increases expression of a TXNIP polypeptide in the cell, increases TXNIP activity in the cell or a combination thereof is selected from the group consisting of glucosamine, an inhibitor of MYC gene expression, an inhibitor of MYC activity and a combination thereof.E193. The method of E192, wherein in the inhibitor of MYC gene expression, the inhibitor of MYC activity, or both is selected from the group consisting of JQ1 , OTX-015, MYCM1-6, EN4, KJPyr9, APTO-253, 10058-F4, Jy-3-094, MYCi361 and a combination thereof.E194. The method of E192 or E193, wherein the inhibitor of MYC gene expression, the inhibitor of MYC activity, or both is JQ1 , OTX-015 or both.E195. A method of selecting a mammalian host cell having one or more of: reduced synthesis of growth inhibitor, reduced synthesis of productivity inhibitor, or improved growth characteristic comprising a) assaying a mammalian host cell for at least one of 1 ) overexpression of a TXNIP gene, 2) overexpression of a TXNIP polypeptide and 3) increased TXNIP activity and b) selecting the host cell comprising at least one of 1) overexpression of the TXNIP gene, 2) overexpression of a TXNIP polypeptide and 3) increased TXNIP activity.E196. The method of E195, the wherein the host cell comprising at least one of 1 ) overexpression of the TXNIP gene, 2) overexpression of a TXNIP polypeptide and 3) increased TXNIP activity has one or more of reduced synthesis of growth inhibitor, reduced synthesis of productivity inhibitor, or improved growth characteristic as compared to synthesis of a growth inhibitor, synthesis of a productivity inhibitor or a growth characteristic of an otherwise identical cell that does not comprise at least one of 1) overexpression of the TXNIP gene, 2)overexpression of TXNIP polypeptide and 3) increased TXNIP activity.E197. The method of E195 or E196, wherein synthesis of the growth inhibitor is reduced by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least100 fold as compared to synthesis of a growth inhibitor by an otherwise identical cell that does not overexpress the TXNIP gene, does not overexpress the TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.E198. The method of any one of E195-E197, further comprising culturing the host cell to produce a first cell culture.E199. The method of E198, further comprising measuring a concentration of the growth inhibitor in the media of the first cell culture on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E200. The method of any one of E195-E199, wherein the growth inhibitor is a cell metabolite.E201. The method of E200, wherein the cell metabolite results from metabolism of amino acids, carbohydrates, fatty acids or a combination thereof.E202. The method of E200 or E201 , wherein the cell metabolite is selected from the group consisting 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate (indole-3-lactate), indolecarboxylic acid (indole-3-carboxylic acid), homocysteine, 2- hydroxybutyric acid, isovalerate, butyrate, isobutyrate, 2-methylbutyrate, formate, lactate, ammonia and a combination thereof.E203. The method of any one of E200-E202, wherein the cell metabolite is lactate.E204. The method of E203, wherein less than 0.01 grams per liter cell media (g / L), 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L of lactate is present in the media of the first cell culture.E205. The method of any one of E195-E204, wherein synthesis of the productivity inhibitor is reduced by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to synthesis of a productivity inhibitor by an otherwise identical cell that does not overexpress the TXNIP gene, does not overexpress the TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.E206. The method of any one of E198-E205, further comprising measuring the concentrationof the productivity inhibitor in the media of the first cell culture on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E207. The method of any one of E195-E206, wherein the productivity inhibitor is a cell metabolite.E208. The method of E207, wherein the cell metabolite results from metabolism of amino acids, carbohydrates, fatty acids or a combination thereof.E209. The method of E207 or E208, wherein the cell metabolite is selected from the group consisting 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate (indole-3-lactate), indolecarboxylic acid (indole-3-carboxylic acid), homocysteine, 2- hydroxybutyric acid, isovalerate, butyrate, isobutyrate, 2-methylbutyrate, formate, lactate, ammonia and a combination thereof.E210. The method of any one of E207-E209, wherein the cell metabolite is lactate.E211. The method of E210, wherein less than 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L of lactate is present in the media of the first cell culture.E212. The method of any one of E195-E211 , wherein the growth characteristic is improved by at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold as compared to a growth characteristic of an otherwise identical cell that does not overexpress the TXNIP gene, does not overexpress the TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.E213. The method of any one of E198-E213, further comprising measuring the growth characteristic of the cell or first cell culture on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, day 15, day 16, day 17 day 18, day 19, day 20 or later of culture.E214. The method of any one of E198-E213, wherein the growth characteristic of the cell or the first cell culture that is improved is selected from the group consisting of cell count (also known as “cell number”), cell viability, viable cell count, cell density, viable cell density, cell metabolism, cell size, and a combination thereof.E215. The method of E198-E214, wherein the growth characteristic of the cell or first cell culture is improved by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to the growth characteristic of a second cell or second cell culture comprising an otherwise identical cell that does not overexpress the TXNIPgene, does not overexpress the TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.E216. The method of E198-E215, wherein the host cell is a CHO cell comprising increased expression of the TXNIP gene, and when cultured, produces a first cell culture that has an increased viable cell density as compared to a viable cell density of a second cell culture comprising an otherwise identical cell that does not overexpress the TXNIP gene.E217. The method of E216, wherein the viable cell density of the first cell culture is increased by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to a viable cell density of a second cell culture comprising an otherwise identical cell that does not overexpress the TXNIP gene.E218. The method of any one of E214-E217, wherein the viable cell density is at least 5 x 105cells per mL cell culture media (cells / mL), 10 x 105cells / mL, 25 x 105cells / mL, 50 x 105cells / mL, 100 x 105cells / mL, 150 x 105cells / mL, 200 x 105cells / mL, 250 x 105cells / mL, 300 x 105cells / mL, 350 x 105cells / mL, 400 x 105cells / mL, 450 x 5005cells / mL, 550 x 105cells / mL, 600 x 105cells / mL or 650 x 105cells / mL.E219. The method of any one of E195-E218, further comprising introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a therapeutic molecule or portion thereof into the host cell.E220. The method of E219, wherein the cell overexpresses the therapeutic molecule or portion thereof.E221. The method of E220, wherein overexpression of the therapeutic molecule or portion thereof is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than expression of the therapeutic molecule by an otherwise identical cell that does not overexpress the TXNIP gene, does not overexpress the TXNIP polypeptide, does not have increased TXNIP activity or a combination thereof.E222. The method of any one of E219-E221 , wherein the therapeutic molecule is a recombinant protein, optionally an antibody and optionally a bispecific antibody.E223. The method of any one of E219-E222, wherein when the cell is cultured in a cell culture, the cell produces at least 0.01 g recombinant protein per L cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1 .2 g / L, 1 .4 g / L, 1 .6 g / L, 1 .8 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L. 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 / g / L, 20 g / L or more.E224. The cell of any one of E1 -E98 or the method of any one of E97-E223, wherein the cell is contacted with a molecule that increases expression of a Mondo A gene, a MondoB gene orboth.

[0245] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.EXAMPLESExample 1 : Experiment to determine the time course expression levels of TXNIP protein in CHO cells that metabolicallv shift or not-shift to lactate consumption in fedbatch cultures

[0246] Methods:

[0247] CHO cell line A and B, and C and D were cultivated in two separate 12-day fedbatch cultures in glass bioreactors with 1 L working volume. Cells were inoculated using a basal medium which is a chemically defined protein free amino acid fortified version of Dulbecco’s modified Eagle’s medium:F12 medium with adjusted levels of vitamins, trace elements, sodium bicarbonate and potassium chloride, and containing polyvinyl alcohol. Starting day 2 or 3, bioreactors were supplemented with feed medium which is a chemically defined, protein-free concentrated medium with amino acid, vitamin, and trace elements at levels 3-3.5-fold higher on average, than those in the basal medium. Feed medium was added at a rate to ensure amino acids and other nutrients are not depleted from the culture. Glucose was adjusted daily to maintain residual levels at 2 g / L or higher. pH at the low end and the high end of the pH deadband was controlled by addition of a carbonate solution and or carbon dioxide sparge, respectively. 80 W / m3power per unit volume (P / V) was used for agitation. Temperature was controlled at 36.5 °C. Glucose, lactate, pH and viable cell densities were measured on a daily basis. Titer was measured on the harvest day. RNA samples were taken at different time points. Gene expression analysis was performed using qPCR method employing SYBR Green assay or RNA-Seq method employing next-gen sequencing. With qPCR, TXNIP expression was presented using DCT against B-ACTIN and reads per kilobase of exon per million reads mapped (RPKM) approach was used for assessing gene expression for RNA-Seq method. Western blot analysis was performed using antibodies against TXNIP and B-ACTIN (a housekeeping gene). Cell pellet and supernatant samples were taken for cell line A and B across different points during the fedbatch cultures. Intracellular metabolomics analysis was also performed on these samples using LC-MS, GC-MS or NMR.

[0248] Results:

[0249] FIG. 1A-1 C shows the growth (FIG. 1A), lactate (FIG. 1 B) and TXNIP levels (FIG. 1 C) in CHO cell line A and B fedbatch cultures. Both of these cell lines were obtained from thesame transfection and produced the same recombinant protein. Cell line A undergoes a metabolic shift to lactate consumption in the later stages of the fedbatch culture whereas cell line B does not undergo a metabolic shift. RNA-Seq analysis shows that TXNIP levels significantly increased over time in cell line A that undergoes a metabolic shift but there was only a marginal increase in TXNIP expression in cell line B that doesn’t undergo a metabolic shift to lactate consumption.

[0250] FIG. 2A-2D show growth, lactate and TXNIP levels in cell line C and D fedbatch cultures. Both these cell lines were obtained from the same transfection and produced the same recombinant protein. In fedbatch culture, both these cell lines undergo metabolic shift to lactate consumption. However, peak lactate was lower, and propensity of lactate shift was higher in cell line C compared to cell line D. qPCR analysis and western blot analysis showed the TXNIP levels were significantly higher in cell line C compared to cell line D across the length of the culture. TXNIP mRNA and protein levels increased over time in both the cell lines.

[0251] The data suggest that there was a correlation between TXNIP mRNA (and protein) levels and metabolic shift to lactate consumption.Example 2: Experiment to determine expression levels of MYC across cell lines that metabolicallv shift to lactate consumption or not.

[0252] Methods: See example 1 methods.

[0253] Results:

[0254] RNA seq data for cell line A and cell line B were analyzed for genes differentially expressed between the two cell lines. MYC gene was observed to be differentially expressed between the two cell lines with higher expression observed in cell line B that doesn’t undergo metabolic shift (FIG. 3). MYC has been reported to inhibit TXNIP expression in the literature.Example 3: Experiment to probe effect of MYC inhibition on TXNIP expression and lactate metabolism in ambrl 5 fedbatch cultures of CHO cells

[0255] Methods:

[0256] JQ1 and OTX-015 are established small molecule inhibitors of MYC protein. CHO cell line B was cultivated in 10-day fedbatch cultures in ambrl 5. Medium, process and sampling details for the fedbatch cultures were similar to those explained in methods of Example 1 . The ambrl 5 vessels were treated with 0.4 micromolar JQ1 , 2.5 micromolar OTX or DMSO (vehicle control) on days 4 and 6. RNA samples were taken at different time points and gene expression analysis was performed using qPCR method employing SYBR Green assay. TXNIP and MYC expression levels are presented using DCT against B-ACTIN.

[0257] Results:

[0258] FIG. 4A-4D show viable cell density, lactate, expression levels of MYC and TXNIP, respectively, for cell line B (non-shifting cell line). Treatment with JQ1 or OTX-15 did not cause any significant growth differences compared to the control (DMSO) condition until day 8. However, the lactate production was reduced in both the treated conditions compared to the control condition. MYC transcripts levels were lower in the both the treated conditions compared to the control condition. TXNIP expression was higher in treated conditions as compared to the control conditions.Example 4: Experiment to probe effect of MYC inhibition on TXNIP expression and lactate metabolism in CHO cell fedbatch cultures executed in 1 L working volume glass bioreactors

[0259] Methods:

[0260] JQ1 is an established small molecule inhibitor of MYC protein. CHO cell line A and B were cultivated in 10-day fedbatch cultures in 1 L working volume glass bioreactors as described in Example 1 . Two bioreactors were set up for each cell line. For cell line A, one bioreactor was treated with JQ1 on days 4 and 5 and the other bioreactor was treated with DMSO (vehicle control) on the same days. For cell line B, one bioreactor was treated with 0.25 micro molar of JQ1 on days 4, 5, 7, 8 and 9, and the other bioreactor was treated with DMSO (vehicle control) on the same days. RNA samples were taken at different time points and gene expression analysis was performed using qPCR method employing SYBR Green assay. TXNIP and MYC expression levels were measured using DCT against B-ACTIN.

[0261] Results:

[0262] Figure 5A-D show viable cell density, lactate, expression levels of MYC and TXNIP, respectively, for cell line A (shifting cell line). For cell line A, treatment with JQ1 (black filled circles) resulted in growth inhibition compared to the non-treated condition (grey filled squares). But interestingly, the treated condition underwent a metabolic shift to lactate consumption immediately after treatment with JQ1 on day 4. Treatment with JQ1 resulted in reduction in MYC transcript level whereas TXNIP expression increased compared to the untreated condition. Figure 5E-5H show viable cell density, lactate, expression levels of MYC and TXNIP, respectively, for cell line B (non-shifting cell line). For cell line B, treatment with JQ1 (black filled circles) led to impairment of growth compared to the control condition (grey filled squares) initially. However, untreated condition subsequently lost viability (data not shown) and experienced a drop in viable cell density, which was not observed in JQ1 treated condition. The drop is viability in the untreated conditions could be explained by ever increasing lactate levels in this culture. Interestingly, in the treated condition, rate of lactate production significantly decreased post treatment with very little accumulation happening.

[0263] Data in the previous example and this example establishes that MYC inhibitioncauses TXNIP induction and reduction in lactate production, or shift to lactate consumption, in CHO cells.Example 5: Experiment to probe the hexosamine pathway metabolic intermediates levels in fedbatch cultures of CHO cell lines that metabolically shift to lactate consumption or do not shift

[0264] Methods:

[0265] See Example 1 methods for fedbatch culture bioreactor set up and execution for cell line A and B. Cell pellet and supernatant samples were taken for cell line A and B across different points during the fedbatch culture. Intracellular and extracellular metabolomics analysis was performed on these samples.

[0266] Results:

[0267] FIG. 6A-6C shows the intracellular levels of hexosamine biosynthesis pathway intermediates including N-acetylglucosamine 6-phosphate, N-acetylglucosamine / N- acetylgalactosamine and N-acetyl-glucosamine 1 -phosphate in fedbatch cultures of CHO cell lines A and B. Levels of these glucosamine-related metabolites were higher in the cell line A as compared to cell line B at most time points across the fedbatch cultures. Data suggests that the shifting cell lines have higher intracellular levels of glucosamine related intermediates than those that don’t shift to lactate consumption. Glucosamine has been reported to induce TXNIP in the literature.Example 6: Experiment to test the effect of glucosamine treatment, independently or in conjunction with MYC inhibitor JQ1 , on TXNIP expression and lactate metabolism

[0268] Methods:

[0269] CHO cell lines B, E and F producing recombinant proteins were cultivated in multiple pH adjusted shake flask fedbatch cultures. Cells were inoculated at 1 x 106cells / mL viable cell densities on day 0 of fedbatch cultures. Medium, process and sampling details for the fedbatch cultures are similar to those explained in the methods of Example 1 . The flasks were incubated in 36.5 °C and 5% CO2 environment and held on a shaking platform agitated at 140 rpm. The pH in the shake flasks was adjusted once or twice daily using a carbonate solution. For each cell line, the individual shake flasks were treated with JQ1 , glucosamine (GlcN), combination of JQ1 and glucosamine (JQ1+GlcN) or DMSO (vehicle control, also referred to as control). For cell line B and E, treatments with glucosamine (10 mM) and / or JQ1 (0.4 micromolar) were performed on days 4, 6 and 8. For cell line F, glucosamine treatments were performed on days 4 (10 mM), 7 (10 mM), 9 (5 mM) and 10 (5 mM), whereas JQ1 treatments was done on day 4 (0.4 micromolar). RNA samples were taken at different time points and gene expression analysis was performed using qPCR method employing SYBR Green assay. For each cell line,TXNIP expression levels are presented as fold change to day 3 levels in the control condition.

[0270] Results:

[0271] FIG. 6A-6C, FIG. 6D-6F, FIG. 6G-6I show viable cell density, lactate profiles and TXNIP gene expression levels in fedbatch cultures of cell lines B, E and F. In case of cell line B, control condition produced significant amount of lactate which resulted in viability drop causing the culture to end on day 6. For cell line B, all three treated conditions, JQ1 , GlcN and JQ1+GlcN, metabolically shifted and consumed lactate to varying levels resulting in sustained viability and viable cell densities. In case of cell line E and F, control condition continuously produced lactate without undergoing a metabolic shift. Akin to cell line B, for cell lines E and F, treatment with JQ1 , GlcN or both resulted in metabolic shift to lactate consumption with varying propensity. Combined treatment of JQ1 and GlcN (JQ1+GlcN) had the most robust lactate shift across all three cell lines. In addition, treated conditions had higher titers (product of interest) compared to the untreated conditions, except the GlcN treated condition for cell line E (date not shown). Further, for the three cell lines, qPCR analysis showed that treatment with GlcN, JQ1 and JQ+GIcN induced TXNIP but to varying levels. JQ1+GlcN treatment resulted in higher TXNIP induction in two of the three cell lines. Data suggests supplementation of JQ1 and / or GlcN to CHO cell fedbatch cultures result in metabolic shift to lactate consumption and concurrent induction of TXNIP.Example 7: Experiment to overexpress mouse TXNIP gene in CHO cells

[0272] Methods:

[0273] Cells, medium, and shake flask cultures

[0274] CHO cell line G producing a proprietary recombinant antibody was used for overexpression of exogenous mouse TXNIP gene. A proprietary internal medium was used for cell culture for inoculation of seed flasks and T-75 flasks for transfection. Transfections were performed in T-75 flasks (Corning, Tewksbury, MA). 1 mg / mL G418 (neomycin, Invivogen, San Diego, CA) was used for selection of transfection pools. Recovered pools were cultivated at 36.5 °C, 5% CO2, and 80% humidity, with 140 rpm shaker speed for shake flask culture. Viable cell densities and viabilities were measured on a ViCell Automated cell counter (Beckman Coulter, Brea, CA). Metabolites were measured on a NovaFlex instrument (Nova Biomedical, Waltham, MA).

[0275] Construction of TXNIP Overexpression Vectors, Transfections

[0276] A plasmid vector for constitutive overexpression of mouse TXNIP gene was generated using a complementary cDNA from the Mammalian Gene Collection, which is commercially available as a glycerol stock from Dharmacon (Lafayette, CO). New England Biolabs OneTaq master mix (New England Biolabs, Ipswich, MA) and primers from IntegratedDNA Technologies (IDT, Coralville, Iowa) were used to amplify the TXNIP coding defining sequence (CDS). TXNIP CDS was cloned into a pcDNA 3.1 / V5-His-TOPO TA Expression vector (Thermo Fisher, Waltham, MA). A 3.1 / V5-His-TOPO-LacZ TA Expression Vector from the same kit was used as a negative control.

[0277] qPCR Analysis

[0278] Relative expression of TXNIP was evaluated using a customized TaqMan Assay with IDT 2x Prime Time qPCR master mix. Primers and a fluorescent probe tagged with a FAM 5’ fluorescent activator and 3’ Iowa Black Quencher were generated using Geneious® software (Geneious, Boston, MA). The forward primer sequence is 5’ CGGACGGGTGATAGTGGAAG 3’. Reverse primer sequence is 5’ ACTGCTGAGACCCTTGCATC 3’. Florescent probe sequence is 5’ AGTTACCCGAGTCAAAGCCG 3’. Primers, probe were purchased from IDT. 5 million cell pellets were flash frozen and extracted using an Rneasy Kit (Qiagen, Hilden, Germany). Reverse transcription was performed using a Superscript III reverse transcription kit (Invitrogen, Thermo Fisher, Waltham, MA) and cDNAs were run on an Applied Biosystems 7500 instrument (Thermo Fisher, Waltham, MA). Double delta cT analysis was used to compare expression of TXNIP with a housekeeping gene, beta actin (B-ACTIN). Primer probes from IDT were as follows, with 5’ FAM activator and 3’ Iowa Black Quencher used probes. Forward primer is 5’ CCTCTATGCCAACACAGTGC 3’. Reverse primer is 5’ GATGTGGATCAGCAAGCAGG 3’. Probe sequence is 5’ CCACCATGTACCCAGGCATT 3’. Endogenous CHO TXNIP specific primers and probe sequences used for qPCR analysis are as follows - Forward primer: 5’ GACATCATTCCTGAAGACCACC 3’, Reverse primer: 5’ GAGGCATGAACTGGAACTC 3’ and the Probe: 5' TAGATGACATGGACGGTGCTCAGG 3'.

[0279] Results:

[0280] FIG.8 shows the plasmids and procedure used for transfection of TXNIP (Topo Txnip) or LACZ (Topo Lac Z, control) gene. Each plasmid was transfected into CHO cell line G in quadruplicates. The resultant transfection pools were selected for growth under antibiotic (G418) selection pressure. FIG. 9A and 9B show the average viable cell density and viability recovery profiles post transfection for the quadruplicate TXNIP and LACZ pools. For the mock transfection condition, cell line G was transfected with transfection reagent mix containing no plasmid DNA. Recovery of viability and viable cell densities was observed in the conditions transfected with TXNIP and LACZ plasmids, but not in the mock transfection condition. qPCR analysis showed that TXNIP pools had higher mouse TXNIP transcript levels compared to the LACZ controls (FIG. 9C), whereas the expression of endogenous CHO TXNIP gene was similar between the two conditions (FIG. 9D).Example 8: Experiment to characterize growth and metabolic phenotype of TXNIP and LACZcell pools in fedbatch cultures

[0281] Methods:

[0282] Quadruplicate TXNIP pools or the LACZ pools were cultivated for 10 days in pH adjusted shake-flask in fedbatch mode. Cells were inoculated at 0.5 x 106cells / mL on day 0 of the fedbatch cultures. Medium used for inoculating the cultures was similar in composition to the one used in Example 1 but had relatively lower concentrations of nutrients. Feed medium used was same as that used in Example 1 . Feed medium was added starting day 2 to ensure there was no depletion of amino acids in the cultures. The flasks were incubated in 36.5eC, 80% humidity and 5% CO2 environment, and held on a shaking platform agitated at 140 rpm. G418 was added every other day to maintain the selection pressure. Glucose, lactate, pH and viable cell densities were measured on a daily basis. pH was adjusted daily or twice a day using a carbonate solution. Glucose was adjusted daily to maintain residual levels at 2 g / L or higher.

[0283] Results:

[0284] FIG. 10A-10C shows the average viable cell density, residual glucose and lactate profiles in TXNIP and LACZ pool fedbatch cultures. TXNIP pools grew to slightly higher viable cell densities compared to the LACZ pools. Residual glucose levels were similar between the TXNIP and LACZ pools, whereas lactate profiles were slightly lower in the TXNIP pools compared to LACZ pools. These data suggest TXNIP pools produce lower lactate on a per cell basis compared to the LACZ control pools.Example 9: Experiment to single cell clone the TXNIP pools and analyze the gene expression, growth and metabolic characteristics is maintenance cultures

[0285] Methods:

[0286] TXNIP pools were plated in Corning 96 well flat bottom plates (Corning, Tewksbury, MA) at 1 cell / well using limited dilution. CD CHO medium (Gibco, Thermo Fisher, Waltham, MA) was used as cultivation medium. Plates were maintained in an incubator at, 36.5 °C, 5% CO2 and 80% humidity until cell colonies were visible to naked eye. Colonies were subsequently transferred to 24 well Corning plates (Corning, Tewksbury, MA). Once the cell population in the 24 well plates was at 60% or above confluence, clones were transferred to the 12 well plates and were cultivated until they reached 60% or above confluence. Subsequently, 125 mL shake flasks were inoculated with clones from 12 well plates and cultured at 36.5 °C, 5% CO2, and on 140 rpm shaking platform in CD CHO with 8 mM glutamine (Gibco, Thermo Fisher, Waltham, MA) until vialing and sampling for RNA. TXNIP gene expression analysis of the clones was performed as described in Example 7. Cell line G (wildtype, WT) and the clones with high or no expression of TXNIP were subsequently thawed and expanded in shake-flaskbatch cultures using CD-CHO medium without glutamine (except the thaw and the first passage). For each passage, cells were at inoculated with ~0.2 - 0.4 x 106cells / mL densities and cultivated for 3 or 4 days. The flasks were incubated in 36.5 °C, 80% humidity and 5% CO2 environment, and held on a shaking platform agitated at 140 rpm. Glucose, lactate, pH and viable cell densities were measured on a daily basis. Specific lactate production (qLac) was calculated and plotted for all the clones to assess the amount of lactate produced on a per cell basis at the end of each passage.

[0287] Results:

[0288] A described in the methods, two TXNIP pools (P1 and P4) were single cell cloned. Several clones were isolated using limited dilution method. RNA was collected from these pools and qPCR was performed to assess the mouse TXNIP levels in these clones. FIG. 1 1 shows the mouse TXNIP expression as a DCT against B-Actin. Lower DCT indicates higher mouse TXNIP expression and vice versa. Isolated clones had a wide variety of mouse TXNIP expression. Clones with no mouse TXNIP expression were also isolated that are not included in the DCT plot in FIG. 1 1 . Clones with higher expression of mouse TXNIP, no expression of mouse TXNIP and wild type cell line were evaluated for specific lactate production (qLac) in maintenance cultures. FIG. 12 shows the specific lactate production on harvest day of each passage. <7 / .acwas lower in the clones with higher mouse TXNIP expression when compared to clones with no mouse TXNIP expression or the wild type cell line. The data suggests TXNIP overexpression reduces lactate production in CHO cells.Example 10: Experiment to characterize phenotype in TXNIP clones in fedbatch cultures.

[0289] Methods:

[0290] Cell line G (wildtype, WT) and the clones with high or no overexpression of TXNIP were expanded as described in the previous example and inoculated in 1 L working volume production bioreactor. WT cell line was cultivated in duplicate bioreactors. An additional WT condition, with treatment with OTX-015 (MYC inhibitor) and GlcN, was included as well (referred to as WT+OTX-015+GLCN). A total concentration of 2.5 mM of OTX-015 was delivered to this condition as bolus addition on day 6. A total concentration of 20mM of GlcN was delivered to this condition via feed starting on day 3 and bolus additions on days 4, 5, and 6. The bioreactor setup and medium used were similar to those described in Example 1 . The cells were inoculated on day 0 at a target viable cell density of 1 .5x106cells / mL. Specific rate of lactate production was calculated using 2 time point method and subsequently, 2 time point moving average was calculated for analysis purposes. Glucose concentrations was maintained at higher levels (> 5g / L) during the later days of the experiment across all the conditions to potentially induce endogenous CHO TXNIP gene.

[0291] Results:

[0292] FIG. 13A-13D show time course profiles of viable cell densities, lactate, glucose and moving average of specific lactate production rates. The three clones with higher overexpression of mouse TXNIP gene (black filled circles; P1 P1 F6, P1 P3F10 and P4P3F4) grew to higher peak cell densities and maintained higher viable cell densities till the end of the experiment, when compared to the WT, WT+OTX-015+GLCN (grey filled diamonds) or the TXNIP clones with no overexpression of mouse TXNIP gene (grey filed squares; P1 P4A7 and P1 P2C1). Duplicate cultures of WT had higher cell densities than the WT with treatment or the clones with no overexpression of mouse TXNIP gene. Lactate was slightly lower in all the three clones with higher overexpression of mouse TXNIP gene in the early stages of the experiment compared to the other conditions. In the later stages of the experiment, two of the three clones with higher overexpression of mouse TXNIP, namely P1 P3F10 and P4P3F4, accumulated significantly lower lactate compared to the WT and clones with no overexpression of mouse TXNIP gene. One of the clones with higher overexpression of mouse TXNIP gene (P1 P1 F6), WT and the clones with no mouse TXNIP expression continued to produce and accumulate lactate through the end of the experiment. This is also reflected in the specific lactate production rates - all the three clones with higher overexpression of mouse TXNIP gene had lower lactate production rates in the initial part of the experiment and the two clones with higher overexpression of TXNIP gene, P1 P3F10 and P4P3F4, had very low specific lactate production rates during the later stages of the experiment. The WT treated condition stopped producing lactate in the later stages of the experiment which is also reflected by very low (or negative) specific lactate production rate in the later stages of the experiment. These data establish that TXNIP overexpression successfully reduces lactate production rates in fedbatch cultures of CHO cells. Moreover, TXNIP overexpression also seems to confer growth advantage in fedbatch cultures.Example 11 : Experiment to characterize phenotype in TXNIP clone in HiPDOG and fedbatch cultures operated at 4-6 g / L or 1-2 g / L residual glucose levels

[0293] Methods:

[0294] Cell line G (wildtype, WT) and the overexpression clone P4P3F4 were expanded and inoculated in AMBr250HT vessels with a working volume of 200mL. There were three conditions for both the WT and the clone P4P3F4: 1) a fedbatch condition with glucose maintained at 4-6 g / L for the duration of the culture, 2) a fedbatch condition with glucose levels maintained between 1-2 g / L for the duration of the culture, and 3) a condition operated with the High-end pH controlled delivery of glucose (HiPDOG) strategy (e.g., W02004 / 104186). The cells were inoculated on day 0 with a target seed density of 1 .0x106cells / mL. The bioreactorset up and medium used was similar to that described in Example 1 . For the fedbatch conditions, the feeding strategy and pH control was similar to that described in Example 1 . For the HiPDOG conditions, the high end of the pH deadband was controlled by addition of the nutrient feed. On day 7 of the culture, the control strategy for the HiPDOG conditions was changed to fedbatch. Post-HiPDOG strategy, glucose was maintained at 2g / L levels. Glucose, lactate, pH and viable cell densities were measured on a daily basis. Titer was measured on days 10, 11 , and 12. Protein samples were collected at different time points and protein expression analysis was performed using the Western Blot method. Western blot analysis was performed using antibodies against TXNIP and B-Actin (a housekeeping gene). Densitometry analysis was performed on the Western Blot images using the Imaged software and employing the Uncalibrated Optical Density (OD) method.

[0295] Results:

[0296] FIG. 14A-14D show the time course profiles of viable cell densities, lactate, glucose, and days 10-12 titers. The P4P3F4 clone grew to higher viable cell densities and produced more titer than its WT counterpart under each set of conditions. Further, the P4P3F4 clone grew to higher densities and produced more titer in the fedbatch 1 -2g / L glucose condition and HiPDOG condition than the fedbatch 4-6g / L glucose conditions. The lactate levels were similar and increased over time with no lactate shift in WT and P4P3F4 clone cultivated in fedbatch 4- 6g / L glucose condition . Interestingly, in the 1 -2g / L glucose conditions, the P4P3F4 clone demonstrated a strong lactate shift whereas the WT did not undergo a metabolic shift to lactate consumption. However, the WT produced lower levels of lactate in the 1 -2g / L glucose condition than the 4-6g / L glucose. Both the P4P3F4 clone and the WT demonstrated strong lactate shifts in the HiPDOG conditions. FIG. 15A-15C show Western Blot analysis on the time course samples for the 4-6g / L glucose, 1 -2g / L glucose, and HiPDOG conditions.

[0297] Western Blot analysis showed that the P4P3F4 clone has higher TXNIP protein expression than the WT for all conditions. FIG. 16A-16C show densitometry analysis of the Western Blots to determine the relative protein concentration for each condition. Densitometry demonstrated that relative TXNIP protein expression is higher in the P4P3F4 clone than in the WT for each condition. However, in the 4-6 g / L glucose condition, the TXNIP protein levels decreased over the time in both the WT and the P4P3F4 clone. Whereas in the 1 -2 g / L glucose condition, the TXNIP expression increased over time for the P4P3F4 clone but remained low for the WT. In HIPDOG condition, the TXNIP protein expression increased over time in the P4P3F4 clone and dropped a bit towards the end of the run whereas the TXNIP protein expression in the WT dropped initially and increased later in the culture. The trend in the WT HiPDOG condition was expected as lower glucose levels during the period when HIPDOG strategy was operational would have reduced TXNIP expression in WT. These data suggest that (1) 4-6 g / L glucose levels seem to negatively impact TXNIP protein level and (2) at 1-2 g / Lglucose levels, high TXNIP protein levels appears to induce metabolic shift to lactate consumption.

[0298] Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein and the claimed invention below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings.

[0299] All references cited herein, including patents, patent applications, papers, textbooks, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0300] The foregoing description and Examples detail certain specific embodiments of the invention and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.

[0301] It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of” are also provided.

[0302] Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.

[0303] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer orgroup of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting. The term “or” when used in the context of a listing of multiple options (e.g. “A, B, or C”) shall be interpreted to include any one or more of the options, unless the context clearly dictates otherwise.

[0304] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

CLAIMSWe claim:1 . A mammalian host cell comprising an exogenous thioredoxin interacting protein (TXNIP) gene.

2. The cell of claim 1 , wherein the cell is a mouse cell, a rat cell, a Chinese Hamster Ovary (CHO) cell or a human cell.

3. The cell of claim 1 or 2, further comprising an exogenous gene encoding a therapeutic molecule or portion thereof.

4. The cell of claim 3, wherein the therapeutic molecule is a recombinant protein.

5. The cell of claim 4, wherein the recombinant protein is selected from the group consisting of an antibody or fragment thereof, nanobody, glycoprotein, growth factor, clotting factor, cytokine, fusion protein, pharmaceutical drug substance, vaccine, enzyme, receptor, hormone, regulatory factor, antigen, binding agent and detectable protein.

6. The cell of any one of claims 1 -5, wherein the TXNIP gene is chromosomally-integrated in a host cell chromosome.

7. The cell of claim 6, wherein the host cell chromosome contains a recombination target site for site-specific integration of the exogenous TXNIP gene in a host cell chromosome.

8. The cell of any one of claims 1 -7, wherein the cell has one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic compared to an otherwise identical cell that does not contain an exogenous TXNIP gene.

9. The cell claim 8, wherein the improved growth characteristic is selected from cell count, cell viability, viable cell count, cell density, viable cell density, cell metabolism, cell size or a combination thereof.

10. The cell of claim 9, wherein the cell metabolism is a metabolic shift by the cell, and optionally wherein the metabolic shift comprises an increased lactate consumption, adecreased lactate production or a combination thereof.11 . The cell of any one of claims 1 -10, wherein the nucleotide sequence encoding the exogenous TXNIP gene encodes a polypeptide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 , comprises a polynucleotide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 2, or both.

12. A host cell culture comprising a host cell of any one of claims 1-11 , wherein the host cell is a CHO cell, and wherein the host cell culture has one or more of 1) an increased viable cell density and 2) exhibits a metabolic shift, as compared to a viable cell density or metabolic shift of a host cell culture comprising an otherwise identical cell that does not comprise an exogenous TXNIP gene.

13. The cell culture of claim 12, wherein the metabolic shift comprises an increased lactate consumption, a decreased lactate production or a combination thereof.

14. The cell culture of claim 13, wherein the increased lactate consumption, the decreased lactate production or combination thereof is detected by measuring lactate concentration in media of the cell culture.

15. The cell culture of claim 14, wherein the lactate concentration in the media of the cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold lower than a lactate concentration in media of a second cell culture comprising an otherwise identical host cell that does not comprise an exogenous TXNIP gene.

16. The method of any one of claims 12-15, wherein the viable cell density of the cell culture is increased by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to a viable cell density of a second cell culture comprising an otherwise identical host cell that does not comprise an exogenous TXNIP gene.

17. A method of selecting a mammalian host cell having one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic, the method comprising: a) assaying a mammalian host cell for at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity; b) selecting the host cell comprising at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity, wherein the host cell comprising at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity has one or more of reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic as compared to synthesis of a growth inhibitor, synthesis of a productivity inhibitor or a growth characteristic of an otherwise identical cell that does not comprise at least one of 1) overexpression of the TXNIP gene, 2) increased copy number of the TXNIP gene and 3) increased TXNIP activity.

18. The method of claim 17, wherein the improved growth characteristic is an increased viable cell density when the host cell is grown in a cell culture.

19. The method of claim 17, wherein the growth inhibitor or productivity inhibitor is a cell metabolite, optionally lactate.

20. A method of preparing a mammalian host cell having one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic, the method comprising: introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP into the host cell, wherein the host cell comprising the exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP has one or more of: reduced synthesis of a growth inhibitor, reduced synthesis of a productivity inhibitor, or an improved growth characteristic compared to an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.21 . The method of claim 20, further comprising introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a therapeutic molecule into the host cell and culturing the host cell to produce a first host cell culture.

22. The method of claim 21 , wherein the amount of therapeutic molecule produced by the first host cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold greater than the amount of therapeutic molecule produced by a second host cell culture comprising an otherwise identical mammalian host cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.

23. The method of claim 21 or 22, wherein the therapeutic molecule is a recombinant proteinand at least 0.01 grams protein per liter cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1 .2 g / L, 1 .4 g / L, 1 .6 g / L, 1 .8 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L. 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L or 20 g / L is produced by the first host cell culture, optionally, wherein the protein is an antibody, and optionally wherein the antibody is a bispecific antibody.

24. The method of any one of claims 21-23, wherein the first host cell culture has one or more of: 1) an increased viable cell density and 2) exhibits a metabolic shift as compared to a viable cell density or metabolic shift of a second host cell culture comprising an otherwise identical mammalian host cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.

25. The method of claim 24, wherein the metabolic shift comprises an increased lactate consumption, a decreased lactate production or a combination thereof.

26. The method of claim 25, wherein the increased lactate consumption, decreased lactate production or combination thereof is detected by measuring lactate concentration in media of the first host cell culture.

27. The method of claim 26, wherein the lactate concentration in the media of the first host cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold lower than a lactate concentration in media of a second host cell culture comprising an otherwise identical mammalian host cell that does not comprise anexogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.

28. The method of claim 26 or 27, wherein the lactate concentration in the first host cell culture media is less than 0.01 grams lactate per liter cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L.

29. The method of any one of claims 22-27, wherein the viable cell density of the first host cell culture is increased by at least 10%, 20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, or 500% as compared to a viable cell density of the second host cell culture comprising an otherwise identical cell that does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP.

30. The method of any one of claims 24-29, wherein the viable cell density of the first host cell culture is at least 5 x 105cells per milliliter cell culture media (cells / mL), 10 x 105cells / mL, 25 x 105cells / mL, 50 x 105cells / mL, 100 x 105cells / mL, 150 x 105cells / mL, 200 x 105cells / mL, 250 x 105cells / mL, 300 x 105cells / mL, 350 x 105cells / mL, 400 x 105cells / mL, 450 x 5005cells / mL, 550 x 105cells / mL, 600 x 105cells / mL, 650 x 105cells / mL, 700 x 105cells / mL, 750 x105cells / mL, 800 x 105cells / mL, 850 x 105cells / mL, 900 x 105cells / mL, 950 x 105cells / mL or 1000 x 105cells / mL31 . The method of any one of claims 17-30, wherein the host cell, the host cell culture or both are contacted with a molecule that increases expression of a TXNIP gene or TXNIP polypeptide.

32. The method of claim 31 , wherein the molecule is glucosamine, a molecule that inhibits expression of the MYC gene, a molecule that inhibits MYC activity or a combination thereof.

33. A method for producing a recombinant therapeutic molecule or portion thereof, the method comprising: a) providing a host cell of any one of claims 1 -1 1 , a host cell culture of any one of claims 112-16 or a host cell selected or prepared by a method of any one of claims 17-32, and b) culturing the host cell or host cell culture under conditions sufficient to produce the recombinant therapeutic molecule.

34. The method of claim 33, further comprising recovering the recombinant therapeutic molecule from the cell or cell culture.

35. The method of any one of claims 20-34, wherein the nucleotide sequence encoding the exogenous TXNIP gene 1) encodes a polypeptide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 , 2) comprises a polynucleotide that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 2, or 3) both.

36. A method for producing a metabolic shift in a host cell, the method comprising1) introducing an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP into the host cell,2) contacting a host cell comprising a TXNIP gene with a molecule that increases expression of a TXNIP gene in the cell, increases expression of a TXNIP polypeptide in the cell, increases TXNIP activity in the cell or a combination thereof, or3) both, and optionally, wherein the host cell is a CHO cell.

37. The method of claim 36, wherein the metabolic shift comprises an increased lactate consumption, a decreased lactate production or a combination thereof.

38. The method of claim 36 or 37, further comprising culturing the host cell in a cell culture, and measuring lactate concentration in media of the host cell culture.

39. The method of 38, wherein the lactate concentration in the media of the host cell culture is at least 1 .5 fold, at least 2 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold at least 100 fold lower than a lactate concentration in media of a second host cell culture comprising an otherwise identical host cell that 1) does not comprise an exogenous nucleic acid molecule comprising a nucleotide sequence encoding TXNIP, 2) is not contacted with a molecule that increases expression of a TXNIP gene in the cell, increases expression of a TXNIP polypeptide in the cell, increases TXNIP activity in the cell or a combination thereof or 3) both.

40. The method of claim 38 or 39, wherein the lactate concentration in the media of the host cell culture is less than 0.01 grams lactate per liter cell culture media (g / L), 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L.41 . The method of any one of claims 36-40, wherein the molecule that increases expression of the TXNIP gene in the cell, increases expression of the TXNIP polypeptide in the cell, increases TXNIP activity in the cell or a combination thereof, is selected from the group consisting of glucosamine, an inhibitor of MYC gene expression, an inhibitor of MYC activity and a combination thereof.

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