Cloned Chinese Hamster Ovary Cells and Their Uses

Abstract

Provided herein are clonal Chinese Hamster Ovary (CHO) cells and their use in increasing cellular productivity in biomanufacturing processes. Specifically, the disclosure provides a method for improving the specific cellular productivity of a polypeptide of interest in a recombinant cell, comprising expressing a nucleic acid encoding the polypeptide of interest in a recombinant cell having a high mitochondrial membrane potential (MMP).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application, filed on November 30, 2022, claims priority under 35 USC § 119(e) to the following U.S. Provisional Application No. 63 / 264,781, filed on December 1, 2021. Each of the above-listed applications is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] Improving recombinant protein production using Chinese Hamster Ovary (CHO) host cells is a major challenge for biopharmaceutical companies. Phenotypic and genotypic variations within the CHO host population are known to contribute to their versatility in growth, metabolism and biosynthetic capabilities (Non-Patent Document 1). Host cell heterogeneity, along with random integration of expression vectors into the host genome, results in recombinant cell lines expressing proteins with various productivity and product quality profiles. Thus, the development of cell lines for biotherapeutic protein production requires extensive screening of heterogeneous transfectant populations to identify and isolate high-producing cells. Obtaining host cell lines that exhibit a more uniform biomanufacturing phenotype would significantly improve the efficiency of the cell line development process. Thus, there is a need in the art for host cell lines with improved biomanufacturing capabilities. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Davies,SLet al.Biotechnol Bioeng 110,260-274,(2013);O'Callaghan,PMet al.Biotechnol Prog 31,(2015) Summary of the Invention [Means for solving the problem]

[0004] The present disclosure relates to a method for improving the specific cell productivity of a polypeptide of interest in a recombinant cell, comprising expressing a nucleic acid encoding the polypeptide of interest in a recombinant cell having a high mitochondrial membrane potential (MMP).

[0005] The present disclosure also relates to a method for improving lactate metabolism in a recombinant cell, comprising expressing a nucleic acid encoding a polypeptide of interest in a recombinant cell having an increased mitochondrial membrane potential (MMP).

[0006] In one embodiment, at least 90% of the cells have a cytometric assay of >10 3 With log MMP fluorescent staining intensity. In one embodiment, the dye used in the flow cytometry analysis is Mito-ID, Rh123, DioC6, JC-1, or tetramethylrhodamine methyl ester (TMRM).

[0007] In another embodiment, the recombinant cell is a Chinese Hamster Ovary (CHO) cell.

[0008] In another aspect, the recombinant cells are comprised in a cell culture. In another aspect, the cell culture is a batch culture, a fed-batch culture, a continuous culture, or a perfusion culture. In another aspect, the cell culture is a fed-batch culture. In another aspect, the recombinant cells are adapted to grow in suspension. In another aspect, the cells are cultured in a bioreactor.

[0009] In one embodiment, the recombinant cell stably expresses the polypeptide of interest. In another embodiment, the polypeptide of interest is an antibody or a soluble receptor. In another embodiment, the polypeptide of interest is an antibody. In another embodiment, the polypeptide of interest is produced at a level of at least 10 pg / cell / day, at least 15 pg / cell / day, at least 20 pg / cell / day, or at least 25 pg / cell / day. In another embodiment, the polypeptide of interest is recovered.

[0010] In one embodiment, the recombinant cells have undergone at least 25, at least 50, at least 75, or at least 100 divisions. In another embodiment, the viability of the recombinant cells is such that at least 90% of the cells have a viability of <10 as determined by flow cytometry. 3 The recombinant cells have increased levels of mGPDH, GAS7, and Mfn2 gene expression or have been engineered to overexpress mGPDH, GAS7, and / or Mfn2, relative to the expression levels in a control population of recombinant cells.

[0011] The present disclosure also relates to a method for improving cell cloning efficiency of a recombinant cell, comprising isolating cells having a high level of mitochondrial membrane potential (MMP) and growing said cells under conditions that promote cell growth. In another embodiment, the recombinant cell is a CHO cell. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 shows the generation of MMP-enriched hosts. Figure 1A shows the enrichment process for high MMP subpopulations using fluorescence-activated cell sorting (FACS). The dotted boxes in the histogram plots represent the sorted populations. Figure 1B shows a bivariate plot of green fluorescence (x-axis) versus orange fluorescence (y-axis) and a univariate histogram plot of orange fluorescence showing the Mito-ID dye staining patterns of standard CHO and MMP-enriched hosts. [Diagram 2]Figure 2 shows the phenotype of MMP-enriched hosts. Figure 2A shows two representative confocal microscopy images of Mito-ID dye stained standard CHO and MMP-enriched hosts, respectively. Orange fluorescence in cells indicates energized mitochondria. Figure 2B shows a histogram plot of mitochondrial mass determined by nonyl acridine orange (NAO) staining followed by flow cytometry analysis. Figure 2C shows flow cytometry analysis of the stability of MMP phenotype in MMP-enriched hosts over several generations. Bivariate plot of green fluorescence (x-axis) versus orange fluorescence (y-axis) and univariate histogram plot of orange fluorescence showing Mito-ID dye staining pattern. PDL: population doubling level. [Diagram 3] Figure 3 shows fed-batch characteristics of stable transfectant pools generated from standard CHO and MMP enriched hosts. Three molecules with different expression difficulties were selected (ETE, DTE1 and DTE2). Figures 3A-C show the integrated viable cell density (IVC) of the pools. Figures 3D-F show the viability of the pools. Figures 3G-I show the recovered titers of the pools. Figures 3J-L show the specific productivity (Qp) of the pools. Data represent the mean ± SD (n=4). *p<0.05 by two-tailed Student's t-test. ETE, easy expression; DTE, difficult expression. [Figure 4] Figure 4 shows metabolic profiles of pools expressing ETE, DTE1 and DTE2 in fed-batch cultures. Figures 4A-C show glucose consumption of the pools and Figures 4D-F show lactate production of the pools. Data represent mean ± SD (n=4). [Diagram 5] FIG. 5 shows representative flow cytometry histogram plots of the MMP status of stable pools from standard CHO and MMP-enriched hosts as determined by Mito-ID staining. [Figure 6]Figure 6 shows characterization of clones derived from standard CHO and MMP enriched hosts expressing difficult-to-express molecules (DTE1 and DTE2). Figure 6A shows colony growth in 384-well plates after 16 days of single cell sorting. Figures 6B-C show recovery titers of clones after 13 days in fed-batch culture. Each data point represents an individual antibody-expressing clone. Figures 6D-E show aggregate levels determined by high molecular weight (HMW) product analysis. Data in D and E represent mean ± SD (n=6). ***p=0.0016, **p=0.004, ***p=0.0088 by two-tailed Student's t-test. [Figure 7] Figure 7A shows a volcano plot showing the expression profile of proteins in standard CHO and MMP-enriched host cells. The dotted line represents the 5% FDR adjusted p-value cutoff for protein quantification. Some of the proteins are labeled using the predicted mouse homolog genes. Figure 7B shows a heat map of differentially expressed proteins plotted using Perseus software. Z-scores (the mean of each column subtracted from each value divided by the standard deviation) were calculated using log2 abundance. Proteins were classified based on their localization and significant functional classes obtained from Ingenuity pathway software (Qiagen). Triplicate data from CHO and MMP-enriched host are shown. [Figure 8] Figure 8 shows Western blot analysis of mitochondrial and ER-associated proteins. Figure 8A shows that whole cell lysates and mitochondrial lysates of standard CHO and MMP-enriched host cells were analyzed for proteins involved in mitochondrial function and high MMP phenotype. Figures 8B-C show that whole cell lysates of stable pools made from standard CHO and MMP-enriched hosts were analyzed for mitochondrial and ER stress proteins. [Figure 9-1]Figure 9 shows a bivariate flow cytometry plot of green fluorescence (x-axis) versus orange fluorescence (y-axis) showing the Mito-ID dye staining pattern of cells grown from 72 wells isolated after the second round of enrichment sorting. Wells containing high MMP cells selected for generation of MMP-enriched hosts are indicated by red dots. [Figure 9-2] Same as above. [Figure 10] FIG. 10 shows the transgene expression pattern of the stable pools as determined by intracellular staining of the light and heavy chains using fluorescently conjugated antibodies. [Figure 11] FIG. 11 shows a scheme for a general TMT-based quantitative proteomic analysis. [Figure 12-1] Figure 12 shows bioreactor fed-batch cell culture performance evaluation for parental CHO clones expressing DTE1 and DTE2 (CDE1a and CDE2a) and MMP enriched clones (MDE1a and MDE2a). Figures 12A-B Titer at day 14. Figures 12C-D Specific productivity (Qp). Figures 12E-F Viable cell density (VCD). Figures 12G-H Viability. Figures 12I-J Glucose consumed. Figures K-L Lactic acid produced. Data represent mean ± SD (n=2). *p<0.05 by two-tailed Student's t-test. [Figure 12-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] This disclosure provides an innovative approach to enrich recombinant host cells with high mitochondrial membrane potential (MMP). Stable transfectant pools and clonal cell lines expressing poorly expressed bispecific molecules generated from MMP-enriched hosts outperformed standard hosts by exhibiting (1) improved fed-batch productivity, (2) improved lactate metabolism, (3) enhanced long-term cell viability in fed-batch cultures, and (4) improved cell cloning efficiency during monoclonal cell line generation. To investigate the underlying mechanisms by which high MMP affects productivity, proteomic analysis was used in conjunction with Western blot validation. MMP-enriched hosts exhibited multifaceted protection against mitochondrial dysfunction and ER stress.

[0014] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used herein, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the specification.

[0015] It should be noted that the term "a" refers to one or more of that entity, for example, "feed medium" is understood to represent one or more feed mediums. Thus, the terms "a," "one or more," and "at least one" may be used interchangeably herein.

[0016] The term "and / or" as used herein should be construed as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0017] Whenever an embodiment is described herein using the term "comprising," it is understood that similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0018] 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 disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0019] Units, prefixes, and symbols are expressed in the format accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure which can be had by reference to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0020] The use of the alternative (e.g., "or") should be understood to mean either, both, or any combination thereof of the alternatives. As used herein, the indefinite article "a" should be understood to refer to "one or more" of the named or listed components.

[0021] The term "about" or "essentially consisting of" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially consisting of" can mean within or more than one standard deviation, as is customary in the art. Alternatively, "about" or "essentially consisting of" can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, these terms can mean values ​​up to an order of magnitude or 5-fold. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially consisting of" should be assumed to be within an acceptable error range for that particular value or composition.

[0022] As used herein, concentration ranges, percentage ranges, ratio ranges, or integer ranges, unless otherwise indicated, should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (such as tenths and hundredths of integers).

[0023] "Mitochondrial membrane potential" or "MMP" is an important indicator of mitochondrial activity as it reflects the processes of electron transport and oxidative phosphorylation, which are the driving forces behind ATP production. Loss of MMP is often associated with early stages of apoptosis. The collapse of MMP coincides with the opening of the mitochondrial permeability transition pore, resulting in the release of cytochrome C into the cytosol, which in turn triggers other downstream events in the apoptotic cascade. Cellular MMP can be detected using fluorescent dyes that fluoresce different colors depending on the membrane potential state.

[0024] The terms "polypeptide" or "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also include amino acid polymers that are modified, either naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other modifying operation, such as conjugation with a labeling component. 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, are also included in the definition. The terms "polypeptide" and "protein" as used herein specifically include antibodies and Fc domain-containing polypeptides (e.g., immunoadhesins).

[0025] As used herein, the term "polypeptide of interest" is used in its broadest sense to include any protein (either natural or recombinant) present in a mixture for which purification is desired. Such proteins of interest include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies), and / or any fusion proteins. In some aspects, a protein of interest refers to any protein that may be produced by the methods described herein. In some aspects, the protein of interest is an antibody. In some aspects, the protein of interest is a recombinant protein.

[0026] The terms "recover," "purify," "separate," or "isolate," as used interchangeably herein, refer to increasing the purity of a protein of interest from a composition or sample that contains the protein of interest and one or more impurities. Typically, the purity of a protein of interest is increased by removing (completely or partially) at least one impurity from the composition.

[0027] As used herein, the term "cultivating" or "cell culturing" refers to the maintenance or growth of recombinant cells in a liquid culture medium under a controlled set of physical conditions.

[0028] As used herein, the term "fed-batch culture" or "fed-batch process" refers to a method of culturing cells in which additional components are provided to the culture at some point after the start of the culturing process. Fed-batch culture can be initiated using a basal medium. A culture medium in which additional components are provided to the culture at some point after the start of the culturing process is a feed medium. A fed-batch culture is typically stopped at some point and the cells and / or components in the medium are harvested and optionally purified.

[0029] As used herein, "perfusion" or "perfusion culture" or "perfusion culture process" refers to the continuous flow of a physiological nutrient solution at a constant rate through or above a population of cells. Because perfusion systems generally involve the retention of cells in a culture unit, perfusion cultures characteristically have a relatively high cell density, but the culture conditions are difficult to maintain and control. In addition, as cells grow to the culture unit and then are retained in the culture unit at high density, the growth rate typically decreases continuously over time, leading to a late exponential phase and even a stationary phase of cell growth. This continuous culture strategy generally involves culturing mammalian cells, e.g., anchorage-independent cells, that express a polypeptide and / or virus of interest during the production phase in a continuous cell culture system.

[0030] The term "productivity" or "specific cell productivity" refers to the amount of a particular protein produced by a defined number of cells in a defined time. Thus, specific productivity is a quantitative measure of the ability of a cell to express / synthesize / produce a protein of interest. In the context of industrial manufacturing, specific productivity is usually expressed as the amount of picograms of protein produced per cell and day ("pg / cell / day" or "pcd").

[0031] A "recombinant cell" is typically produced by transfecting recombinant DNA into a host cell. Typically, recombinant host cells useful in the methods of the present invention are mammalian cells. In one embodiment, the recombinant host cell is a Chinese Hamster Ovary (CHO) cell.

[0032] "Antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL comprises three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. In one embodiment, the antibody is a full-length antibody.

[0033] Immunoglobulins may be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, IgD, IgE, and IgM. IgG subclasses are also well known to those skilled in the art, including, but not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies may be humanized by recombinant methods to reduce their immunogenicity in humans. The term "antibody" may include multivalent antibodies (e.g., trivalent antibodies) capable of binding more than two antigens. Trivalent antibodies are IgG-type bispecific antibodies composed of two conventional Fab arms fused to one asymmetric third Fab-sized binding module via a flexible linker peptide. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain fused to CH3 with a "knob" mutation and the variable region of the light chain fused to CH3 with a matching "hole". The hinge region does not contain a disulfide bond to facilitate antigen access to the third binding site. Unless expressly stated and unless the context dictates otherwise, the term "antibody" includes monospecific, bispecific or multispecific antibodies, as well as single chain antibodies.

[0034] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked. Amino acid sequences that are normally present in separate proteins can be brought together in a fusion polypeptide, or amino acid sequences that are normally present in the same protein can be placed in a new arrangement in a fusion polypeptide, such as a fusion of an Ig Fc domain with domain VIII of the present disclosure. Fusion proteins are created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. Chimeric proteins can further comprise a second amino acid sequence associated with the first amino acid by a covalent, non-peptide or non-covalent bond.

[0035] As used herein, "culturing" refers to growing one or more cells in vitro under defined or controlled conditions. Examples of culture conditions that may be defined include temperature, gas mixture, time, and media formulation.

[0036] As used herein, the term "expression" or "expressing" is used to refer to transcription and translation occurring within a cell. The level of expression of a product gene in a host cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by the product gene produced by the cell, or both. In some aspects, relative expression refers to the amount of mRNA or protein expressed in a cell compared to a control cell. For example, increased expression can refer to an increase in gene / protein expression in a recombinant cell compared to a non-transformed cell. In some embodiments, increased expression requires at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold gene / protein expression in a recombinant cell compared to a non-transformed control cell.

[0037] Various aspects of the disclosure are described in further detail in the following subsections.

[0038] II. High MMP recombinant cells The present disclosure provides a highly effective approach to increase the specific cell productivity of a polypeptide of interest in a recombinant cell by using cells with high levels of MMP. The present disclosure also provides a highly effective approach to improve lactate metabolism in a recombinant cell by using cells with high levels of MMP.

[0039] In one aspect, the present disclosure provides a method for isolating host cell subpopulations that exhibit superior capacity for protein production. In one aspect, CHO cells were stained with MMP-specific fluorescent dyes and enriched for high MMP using fluorescence-activated cell sorting (FACS). In one aspect, the dye is Mito-ID, Rh123, DioC6, JC-1, or tetramethylrhodamine methyl ester (TMRM). In another embodiment, the dye is Mito-ID. The high MMP cells were found to have significantly improved growth characteristics, titers, lactate metabolism, and subcloning performance compared to non-clonal pools.

[0040] In one embodiment, the pool of high MMP recombinant cells is substantially homogenous with respect to MMP. In one embodiment, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells express a minimal MMP. In another embodiment, at least 90% of the high MMP cells express at least 10 minimal MMPs as determined by flow cytometry. 3 , at least 10 4 , or at least 10 5 The MMP fluorescence intensity is log

[0041] In one embodiment, the pool of low MMP recombinant cells is substantially homogenous with respect to MMP. In one embodiment, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells express a minimal MMP. In another embodiment, at least 90% of the low MMP cells have a minimum MMP expression level of <10 as determined by flow cytometry. 3The MMP fluorescence intensity is log

[0042] III. Protein of interest In some embodiments, the methods disclosed herein can be applied to any protein product (e.g., a protein of interest). In one embodiment, the protein is a therapeutic protein. In some embodiments, the therapeutic protein is selected from an antibody or antigen-binding fragment thereof, an Fc fusion protein, an anticoagulant, a blood clotting factor, an engineered protein scaffold, an enzyme, a growth factor, a hormone, an interferon, an interleukin, a receptor, and a thrombolytic agent. In some embodiments, the protein product is an antibody or an antigen-binding fragment thereof. In some embodiments, the protein is a recombinant protein.

[0043] In other embodiments, the protein of interest is produced in a host cell. In some embodiments, the protein of interest is produced in a culture comprising mammalian cells. In some embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), monkey kidney fibroblast cells (COS-7), Madin-Darby bovine kidney cells (MDBK), or any combination thereof. In some embodiments, the starting mixture can be a harvested cell culture fluid, a cell culture supernatant, a conditioned cell culture supernatant, a cell lysate, and a clarified bulk.

[0044] In some aspects, the protein produced by the methods described herein is an antibody. Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In some aspects, the antibodies described herein refer to polyclonal antibody populations. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In some aspects, the antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In one aspect, the antibody is a humanized monoclonal antibody. In some aspects, the antibody is a human monoclonal antibody, preferably an immunoglobulin. In some aspects, the antibodies described herein are IgG1 or IgG4 antibodies.

[0045] The growing demand for therapeutic proteins continues to drive the development of technologies to increase the production of high-quality recombinant proteins in CHO expression systems. Although the yield of CHO cell-based manufacturing processes has been significantly improved in recent years, the ever-expanding competitive market demands still require cells to be made more productive and grown at higher cell densities in bioreactors. In this disclosure, an innovative approach based on the selection of cell populations with high MMP has led to the establishment of novel hosts that inherently exhibit superior attributes for biomanufacturing.

[0046] In certain aspects, the MMP-enriched host demonstrated significantly improved and consistent production capabilities for the three model antibodies examined. In some aspects, increased productivity with higher IVC, enhanced viability and improved lactate metabolism was observed in pools generated from the MMP-enriched host. Furthermore, clones isolated from pools derived from the MMP host demonstrated improved growth in 384-well plates, enrichment of high producing populations and improved bioreactor performance. Furthermore, the high MMP phenotype of the host cell line was observed to be retained in the recombinant production pool indicating that the MMP trait is stable and heritable. Thus, the MMP-enriched host provides a unique tool to generate highly productive, stable and homogenous pool populations that allow for enhanced early-stage material supply for preclinical toxicology testing and process development activities, thereby accelerating the timeline to the clinic. The increased volumetric and specific productivity of two industrially relevant DTE molecules along with their improved lactate profiles demonstrated by the MMP clones in the bioreactor suggest that the MMP-enriched host is better suited to address the challenges of developability and manufacturing of DTE biotherapeutics. Moreover, given these significant improvements in cell performance, this host serves as a cost-effective and high-throughput tool for the cell line development process to produce recombinant proteins in various molecular formats.

[0047] Mitochondria play a dominant role in energy production through the oxidative phosphorylation process of the electron transport chain present in the mitochondrial membrane, and the functional importance of MMP is multifaceted. In general, MMP is an indicator of mitochondrial function and energy metabolism. Apart from being an essential component in the process of energy storage during oxidative phosphorylation, MMP plays an important role in mitochondrial homeostasis through the selective removal of dysfunctional mitochondria. It is also the driving force for the transport of ions (other than H+) and proteins required for healthy mitochondrial function. In addition, MMP provides the driving force for ATP synthesis in mitochondria. Due to their enormous energy demands, productive cells require enhanced mitochondrial function to meet their energy demands. At high MMP, the mitochondrial respiratory chain becomes a significant source of reactive oxygen species production, and maintaining excessively high mitochondrial MMP is known to be harmful to mitochondria and therefore cells, but no harmful effects of high MMP on cell health or productive performance were observed.

[0048] A comparative proteomic analysis of MMP-enriched and standard CHO hosts was also performed. After determining the CHO mouse protein homologs, three pathways regulated in the high-MMP phenotype were identified, and the key proteins involved in these pathways were validated by Western blot analysis. Here, MMP-enriched hosts upregulated the expression of Mfn2, PINK1, and p-Parkin proteins. Mfn2 is known to play multiple critical roles in mitochondrial function, suggesting its influence on mitochondrial homeostasis. Mfn2 has been widely linked to mitochondrial quality control, mainly due to its key role in mitophagy, with PINK1 and Parkin being central to the surveillance mechanism. In this pathway, PINK1 accumulates in defective mitochondria and triggers the translocation of Parkin from the cytosol, mediating the removal of damaged mitochondria. Interestingly, proteomic analysis showed elevated levels of GAS7 in MMP-enriched hosts and transfectant pools, suggesting novel functional importance of these proteins in CHO cells not previously reported. Considerable evidence suggests that Mfn2 depletion leads to reduced MMP, increased mitochondrial proton leakage and impaired fatty acid metabolism and oxidative phosphorylation. Moreover, Mfn2 forms a complex that can tether mitochondria to the ER, a structural feature essential for mitochondrial energy metabolism, maintenance of intracellular calcium homeostasis and regulation of ER stress response. Mfn2 ablation has been shown to induce ER stress in various models, from mouse tissues to Drosophila. In particular, induction of unfolded protein response (UPR) mediators in Mfn2-deficient mouse embryonic fibroblasts under basal or ER stress conditions, and the role of Mfn2 as an upstream modulator of PERK have been reported. In this disclosure, we observed reduced levels of PERK and chaperone proteins BiP and PDI in MMP host-derived pools, and showed that higher levels of Mfn2 expression are consistent with the maintenance of ER homeostasis with good UPR activation and ER stress relief, which most likely contributes to increased productivity of pools and clones, especially for difficult-to-express proteins.Thus, in one aspect, the MMP-enriched host has multifaceted protection against mitochondrial dysfunction and ER stress, thereby providing a more favorable intracellular environment for protein production than the standard CHO host.

[0049] In one aspect, mitochondrial GPDH, an essential component of the mitochondrial respiratory chain and glycerophosphate (GP) shuttle, was upregulated in MMP-enriched host and transfectant pools. Although the specific role of mGPDH in CHO cells has not been defined, its function at the crossroads of glycolysis, oxidative phosphorylation and fatty acid metabolism in mammalian tissues is known. As a rate-limiting component of the glycerophosphate shuttle, mGPDH links mitochondrial and cytosolic processes and plays a key role in cellular bioenergetics. Recent findings suggest a role for mGPDH in regulating cell proliferation and mitochondrial metabolism in various types of cancer, where mGPDH overexpression was found to be associated with increased proliferation and oxidative phosphorylation rates. Thus, in one aspect, the high mGPDH phenotype of MMP-enriched host contributes to increased IVCs in fed-batch cultures and enhanced viability of transfectant pools. The consistency of protein markers observed between MMP-enriched host cells and transfectant pools indicates that FACS-mediated enrichment enabled high MMP hosts to achieve uniformity and phenotypic stability of the high MMP phenotype.

[0050] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, it is for illustrative purposes only and is not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. EXAMPLES

[0051] Cell culture and stable transfection A suspension-adapted proprietary CHO cell line derived from CHO-K1 and glutamine synthetase (GS) selection systems was used. Stable transfectant pools were generated by nucleofection of linearized expression plasmids, then selected and maintained in proprietary medium supplemented with 75 μM methionine sulfoximine (MSX, Sigma-Aldrich, MO) and 50 mg / L dextran sulfate (Sigma-Aldrich). Suspension cell cultures were grown at 120 rpm on an orbital shaking platform in a humidified incubator set at 37°C and 6% CO2. Cells were passaged every 3-4 days. Measurement of viable cell density and viability was achieved using trypan blue and a ViCell automated cell counter (Beckman Coulter, CA).

[0052] Fed-batch culture Antibody production was assessed by fed-batch culture in 125 mL Erlenmeyer flasks or 96DW plates. Production cultures were grown for 14 days at 35.5°C in a humidified 6% CO2 atmosphere unless otherwise stated. Shaking speed was maintained at 120 rpm for flasks and 350 rpm for 96DW. Cell density and viability were monitored during culture in flasks but not in 96DW cultures. Proprietary feed was added to production cultures on days 3, 5, 7, 9, 11 and 13. Antibody titers in culture supernatants were determined using a Protein A biosensor on an Octet QK384 (Pall ForteBio, Fremont, CA) for 96DW and by HPLC (Agilent Technologies, CA) for flask cultures.

[0053] Fed-batch cultivation in bioreactors Production cells were cultivated in duplicate 3 L glass stirred tank bioreactors in fed-batch mode. Cells were cultivated under conditions representative of a large-scale manufacturing process. Temperature of 35.5°C, agitation speed of 300 rpm, dissolved oxygen of 50% and pH of 7.0±0.1 were monitored and regulated using DASGIP controller software (Eppendorf, Germany). Proprietary feeds were added to each bioreactor periodically during operation. Fresh cell samples were collected daily for subsequent process analysis and biochemical assays. Offline pH, gases (pCO2 and pO2), lactate, and glucose levels were analyzed using a RAPIDPOINT 500 blood gas analyzer (Siemens, PA). Viable cell density and cell viability were determined using ViCell. Osmolality and ammonium were measured using a Flex2 (Nova Biomedical, MA). Antibody levels from each bioreactor were quantified using Protein A binding on HPLC.

[0054] Flow cytometric analysis of mitochondrial membrane potential Cellular MMP status was analyzed using the dual-fluorescence dye Mito-ID (Enzo Life Sciences, NY). Cells were stained with Mito-ID dye according to the manufacturer's instructions. Briefly, 1x10 cells were cultured by centrifugation at 300xg for 5 min. 6 Cells were harvested, washed with 1xMito-ID assay solution, and diluted to 1x10 6 The cells were resuspended in Mito-ID detection reagent at a concentration of 1000 cells / mL. The cells were stained for 30 minutes at room temperature in the dark and analyzed using a BD Symphony cytometer (BD Biosciences, CA). A 488 nm laser was used for excitation, and 525 / 50 and 582 / 15 filters were used to acquire green and orange emission fluorescence, respectively. MMP was analyzed using bivariate plots of green versus orange fluorescence, as well as univariate histogram plots of orange fluorescence. The orange fluorescent population represents energized cells with active MMP. Data analysis was performed using FlowJo software (Tree Star, Inc., OR).

[0055] Cell sorting MMP-based bulk cell sorting for host enrichment and single cell deposition for recombinant clone generation were performed using an Influx cell sorter (BD Biosciences) as previously described. 21 For bulk sorting, 5x10 7 Cells were harvested by centrifugation. Cells were washed and stained with Mito-ID dye as described above. Based on orange fluorescence intensity, 2x10 cells from gated fractions were 6 10 cells were placed into a 5 mL collection tube containing culture medium. Sorted cells were centrifuged, resuspended in 10 mL fresh culture medium, and seeded into a T-75 flask. After subculture and expansion, sorted cells were stained with Mito-ID, and subpopulations showing peak intensity of MMP staining were isolated using FACS by depositing 5 cells per well into individual wells of a 96-well plate. For single cell cloning, 1x10 cells were isolated by depositing 1 cell per well into individual wells of a 384-well plate containing conditioned medium. 6 All plates were incubated at 37° C. in a humidified atmosphere of 6% CO2 for proliferation.

[0056] Colony growth measurement Confluence per well of 384-well plates was measured using a Cellavista imager (Synentec, Germany) and results were analyzed using Nyone software (Synentec, Germany).

[0057] Aggregate analysis The fed-batch product was captured by an analytical Protein A column (POROS A, Thermo Fisher Scientific, MA) using an Agilent 1260 HPLC (Agilent, CA) equipped with a fraction collector. PBS was used for the capture step and acidified PBS (with 0.1% phosphoric acid) was used for elution. The purified bispecific antibody was then analyzed for aggregation levels by size-exclusion chromatography (SEC) using an ACQUITY UPLC SEC-200 column (Wasters, MA) on the same Agilent 1260 HPLC. The SEC mobile phase was 0.1 M sodium phosphate, 0.1 M sodium sulfate pH 6.8. The flow rate was set at 0.3 mL / min and the separation was monitored at 280 nm.

[0058] Determination of mitochondrial mass Mitochondrial mass was determined using nonyl acridine orange (NAO, Thermo Fisher Scientific, MA), a dye that binds to the inner mitochondrial membrane independent of membrane potential. Cells were cultured at 1x10 6 Cells were incubated with 500 nM NAO at a concentration of cells / mL for 10 min at room temperature in the dark, washed twice with cold phosphate-buffered saline (PBS), and immediately analyzed on a BD Symphony cytometer using a 488 nm laser for excitation and a 525 / 50 filter for emission. Data analysis was performed using FlowJo software.

[0059] Confocal microscopy All fluorescent images were taken with an LSM880 Airyscanner confocal microscope (Carl Zeiss Inc., Germany). Using a 40x1.2NA water objective, z-stack images of Mito-ID stained cells were acquired from 30 optical sections spaced 0.4 μm apart. Track 1 used a 488 nm laser with a 495-550 nm emission filter, and track 2 used a 561 nm laser with a 570-620 nm emission filter.

[0060] Intracellular staining for antibody expression levels Intracellular expression of heavy and light chains of antibody molecules was determined by staining cells with fluorescently labeled antibodies specific for the heavy or light chain. Briefly, cells were centrifuged, washed with FACS buffer (1% fetal bovine serum in PBS) and fixed with Fixation Medium A (Thermo Fisher Scientific) for 15 min at room temperature. Cells were then washed with FACS buffer and stained with a staining solution consisting of goat anti-human IgG (Fc)-Alexa Fluor 488 (Thermo Fisher Scientific) and goat anti-human κ-APC (Biolegend, CA) in Permeabilization Medium B (Thermo Fisher Scientific) for 15 min at room temperature. Stained cells were washed and resuspended in FACS buffer before being analyzed on a BD Symphony cytometer for APC and AF488 double positive populations. Data analysis was performed using FlowJo software.

[0061] Western blotting Mitochondria were isolated using the Mitochondria Isolation Kit (ThermoFisher Scientific, MA) according to the manufacturer's instructions. Protein extracts were prepared by lysing whole cells and isolated mitochondria in RIPA buffer (ThermoFisher) containing Halt protease and phosphatase inhibitor cocktail (ThermoFisher), followed by measurement of protein concentration using the Pierce BCA Assay Kit (ThermoFisher). Western blot analysis was performed using a WES system (Protein Simple, CA) with a separation module of 12-230 kDa (25-well cartridge). All samples were diluted to a final loading concentration of 0.4 μg / μl, and equal amounts of total protein (2 μg) were loaded into each sample. Instrument settings included 25 min separation at 375 V, 30 min blocking, 30 min incubation of primary and secondary antibodies each, and 15 min chemiluminescence detection. Primary antibodies used were rabbit anti-GAS7, PINK1, β-actin (Abcam, MA), Mfn2, mGPDH (ThermoFisher), phospho-Mfn2 (EMD Millipore, MA), phospho-Ser65-Parkin (Biorbyt, MO), PERK, BiP, PDI (Cell Signaling Technology, MA) and mouse anti-SDHA (Abcam). HRP-conjugated goat anti-rabbit and goat anti-mouse (Abcam) were used as secondary antibodies. β-actin and SDHA (succinate dehydrogenase subunit A) were used as loading controls.

[0062] statistical analysis Data are presented as mean ± standard deviation (SD). Comparison of mean differences between groups was performed by unpaired two-tailed Student's t-test unless otherwise stated. A probability level of p<0.05 was considered statistically significant.

[0063] Proteomic analysis Proteomic sample preparation: CHO and MMP-enriched host cells were lysed and trypsin digested using S-TRAP. Peptides were labeled with 6-plex TMT (Thermo Fisher Scientific) according to the manufacturer's instructions (Figure 11). LC-MS / MS analysis: Data-dependent acquisition (DDA) LC-MS / MS analysis was performed on a Q-Exactive HF-X mass spectrometer. Tandem mass spectrometry data were searched against the Uniprot Cricetulus griseus database (Oct 2020) containing 56,400 protein sequences including common laboratory contaminants using the Mascot 2.7.0.0 search engine (Matrixscience.com) integrated into the Proteome Discoverer (v2.4, Thermo scientific) software.

[0064] Bioinformatics analysis Raw protein abundance values ​​were normalized to the median intensity of each TMT channel. Differential abundance analysis was performed using the Limma Bioconductor package (R version 3.6.0). Volcano plots were generated using the ggplot2 R package (R version 3.6.0). Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA) software (Qiagen).

[0065] Sequence Alignment and Machine Learning All protein sequences in the Chinese hamster proteome were aligned against all protein sequences in the mouse proteome using the pairwise2 module from the Biopython package (Python version 3.7.4). A logistic regression model was developed to predict functional homology based on percent sequence identity. The model was trained and cross-validated using the scikit-learn Python module (Python version 3.7.4). The trained model was used to assign homologs to each CHO protein.

[0066] Example 1 - Generation of novel CHO host cell lines by MMP enrichment Utilizing a simple flow cytometry staining method to monitor MMP in live cells, high MMP subpopulations from CHO host cells were isolated and stained with the MMP dye, Mito-ID. To ensure stable enrichment of the host population exhibiting high MMP, two rounds of sorting by FACS were performed (Figure 1). In the first step, the subpopulations exhibiting peak intensity of MMP staining were isolated by bulk sorting and the resulting cells were subcultured for 2 weeks. After recovery, evaluation of the MMP capacity of the sorted cells showed partial enrichment of the high MMP population (68% in the sorted cells compared to 29% in the original host cells, Figure 1A). A second round of sorting was performed in 96-well plates. As previously described, the subpopulations exhibiting peak intensity of MMP staining were gated and isolated by depositing 5 cells per well in a 96-well plate (Figure 1A). After 2 weeks, 72 wells with colonies that reached >20% confluence were scaled up into wells of a 96-deep-well (96DW) plate and grown for 1 week. Evaluation of 72 cultures showed various MMP status ranging from 40% to 96% (Figure 9). Cultures showing >92% homogeneity of high MMP phenotype (Figure 9) were pooled together to generate MMP-enriched host cells (Figure 1B). Confocal microscopy imaging of Mito-ID stained standard CHO and MMP-enriched hosts validated their respective MMP phenotypes (Figure 2A). To further determine whether MMP enrichment was due to differences in mitochondrial numbers within each cell, standard CHO and MMP-enriched hosts were stained with nonyl acridine orange (NAO) dye to measure mitochondrial mass. Flow cytometry analysis revealed no difference in mitochondrial mass per cell between the two host types (Figure 2B).

[0067] To determine the stability of the high MMP trait, enriched hosts were serially expanded for 109 generations (population doubling level, PDL) and their MMP status was assessed using Mito-ID staining. The enriched population maintained a uniform high MMP phenotype similar to that of the original pool of enriched colonies even after extended culture (99% at 0 PDL to 95% at 109 PDL, Figure 2C), suggesting that the enrichment strategy is essentially effective in generating phenotypically stable host cell lines. Interestingly, although it has been well documented that the inherent plasticity of the CHO nuclear and mitochondrial genomes results in continuous genetic and phenotypic drift in cultured CHO cells carrying recombinant production burdens, it may be possible that host cells without production burdens have a more stable genome and are therefore less susceptible to phenotypic drift.

[0068] Example 2 - Superior performance of MMP-enriched hosts in stable transfectant pools To evaluate the performance of the enrichment hosts, standard CHO and MMP enrichment hosts were transfected with expression plasmids encoding three different molecules: one easily expressed mAb (ETE) and two poorly expressed bispecific antibodies (DTE1 and DTE2). The resulting pools of stable transfectants were evaluated for fed-batch productivity in shake flasks. For all three molecules, the MMP pools showed higher integral viable cells (IVC) than the standard CHO pools (Figure 3A-C) and maintained higher viability (Figure 3D-F). In the ETE, the MMP pools had a 1.9-fold increase in final titer compared to the CHO pools (Figure 3G; 2.13 vs. 4.12 g / L, p=0.04). The MMP pool also showed a 2.2- and 2.1-fold increase in titer for DTE1 (Figure 3H; 0.58 vs. 1.26 g / L, p<0.0001) and DTE2 (Figure 3I; 1.43 vs. 3.01 g / L, p=0.0005), respectively, compared to the CHO pool. Specific productivity of ETE and DTE1 remained unchanged between the CHO and MMP pools (Figures 3J and 3K), but a significant increase in specific productivity was observed in the DTE2 pool derived from the MMP-enriched host (Figure 3L, 11.9 vs. 20.5 pg / cell / day, p=0.0034).

[0069] To determine the turnover during the fed-batch process, we measured the glucose consumption and lactate production (per cell per day) of the pools throughout the culture period. While glucose consumption was comparable between the MMP and CHO pools for all three molecules (Figure 4A-C), lactate production was found to be lower in the MMP pool cultures than the corresponding CHO pools (Figure 4D-F), suggesting an association between higher mitochondrial activity and improved lactate metabolism. Previously, a positive correlation between high mitochondrial membrane potential and lactate consumption rate in CHO cells was described, pointing to a central role of mitochondria for lactate metabolism.

[0070] Intracellular heavy chain (HC) and light chain (LC) protein expression in the pools was investigated on the day of the last passage before the fed-batch process to assess whether differences in the percentage distribution of the expression populations could be the cause of the observed productivity differences between the two hosts. For ETE, the analysis revealed similar expression of HC and LC in both hosts, with 95% HC+LC+ for CHO and 96% for the MMP-enriched host (Figure 10). For DTE1, the CHO host had a slightly smaller HC+LC+ population compared to the MMP-enriched host (Figure 10, 83% vs. 92%), and for DTE2, both hosts showed similar expression of HC and LC, with 94% HC+LC+ for CHO (Figure 10) and 97% for the MMP-enriched host. Thus, the significant differences in productivity observed between the two hosts are not due to differences in the homogeneity of the expression populations. Furthermore, comparison of the MMP status of pools generated from the CHO host and the MMP-enriched host revealed that the high-MMP phenotype of the host was robustly maintained in the stable transfectant pool (Figure 5), thereby further confirming the phenotypic stability of the MMP-enriched host.

[0071] Example 3 - Evaluation of MMP-enriched hosts in the cell line development process The generation of clonal manufacturing cell lines is a critical step to ensure reproducible product quality of biopharmaceuticals. To determine the suitability and applicability of the novel MMP-enriched hosts for successful biomanufacturing, the performance of clones isolated from stable pools was evaluated with a focus on difficult-to-express proteins, since any improvement has a greater potential impact and benefit for production. First, FACS was used to deposit single cells into 384-well plates from pools of DTE1 and DTE2 transfectants generated from standard CHO and MMP-enriched hosts. Clones were then expanded and evaluated in an automated small-scale productivity and product quality screen in a 96DW format. A significant improvement was observed in the 384-well growth of clones isolated from the MMP-enriched pools of both molecules when compared to the standard CHO host (16% vs. 29% for DTE1 and 16% vs. 26% for DTE2) (Figure 6A). Furthermore, 96DW titer evaluation revealed a significant enrichment of high producing clones for DTE1 (p=0.0016) and DTE2 (p=0.0088) made from MMP enriched host (Figures 6B and 6C). Purified product quality analysis of fed-batch culture supernatants revealed a significant decrease (p=0.004) in the content of high molecular weight (HMW) species in DTE2 clones made from MMP enriched host, with no difference observed in DTE1 clones (Figures 6D and 6E). Taken together, the results suggest that MMP enriched host exceeds standard CHO host in overall performance.

[0072] Example 4 - Proteomic Analysis To establish a comprehensive understanding of the distinct phenotypes of standard CHO and MMP-enriched hosts, their subcellular proteomic profiles were compared. Quantitative proteomics using 24 LC-MS / MS analyses derived from fractions of multiplexed tandem mass tag (TMT)-labeled protein digests of CHO and MMP-enriched cells led to the identification of nearly 8,400 protein groups, one of the largest numbers of CHO cell protein profiles reported to date. Of the identified proteins, 90% were quantified using isobaric labeling methods. One challenge encountered was that the Chinese hamster proteome is not as well annotated as the proteomes of model organisms. Therefore, to improve annotation, proteins were matched in the Chinese hamster and mouse proteomes by their UniProt protein descriptions. For proteins that could not be matched, the percent sequence identity of the CHO-mouse protein pairs was calculated and a logistic regression model was developed to distinguish homologs from non-homologs. The model was validated by 10-fold cross-validation, and the model achieved an accuracy of over 97% on both the training and validation datasets.

[0073] Abundance levels of proteins involved in mitochondrial function were analyzed and compared between standard CHO and MMP-enriched hosts. Biological datasets were compared to calculate "fold change" and significance by ANOVA, and results were graphed as volcano plots (Figure 7A). Here, proteins were considered differentially abundant if their fold change was greater than 1.5 and their adjusted p-value was less than 0.05. Overall, 12 proteins were significantly upregulated and 44 proteins were significantly downregulated (at least 1.5-fold) in MMP-enriched cells compared to standard CHO cells (Figure 7A). Biological functions associated with differentially expressed proteins were determined using Ingenuity pathway functional analysis features (Figure 7B). Proteomic datasets from each host type were further annotated using Uniprot GO annotations, including cellular localization and molecular and biological functions. Nearly 1000 mitochondria-associated proteins were found. Ingenuity pathway analysis (IPA) revealed several of these proteins involved in regulating transmembrane potential, depolarization, maintaining morphology, mitochondrial organization and fusion. A heatmap of differentially expressed proteins from this subset is shown in Figure 7B.

[0074] Selected findings of the proteomic assay were confirmed by Western blotting analysis focusing on three pathways, namely the Mfn2 (mitofusin 2), GAS7 (growth arrest specific protein 7) and mGPDH (glycerol-3-phosphate dehydrogenase 2) (GPD2) pathways, which are involved in mitochondrial function and were found to be upregulated in MMP-enriched hosts (Figure 8A) and transfectant pools (Figure 8B). Mfn2 plays multiple roles in maintaining a healthy mitochondrial network and thus ensuring proper energetic and metabolic cellular performance. Mfn2 maintains mitochondrial quality control by mediating mitochondrial fusion, regulating physical contacts between mitochondria and the ER, and removing damaged mitochondria via PINK1 / p-Parkin-mediated mitophagy. Furthermore, GAS7 is known to regulate PINK1 function in regulating mitochondrial dynamics and metabolism. Indeed, increased expression of p-Mfn2, PINK1 and p-Parkin was observed along with Mfn2 and GAS7 in MMP-enriched hosts. Furthermore, several studies have shown the role of Mfn2 as an ER stress regulatory protein and an upstream modulator of PERK. Ablation of Mfn2 in various cell types resulted in upregulation of ER chaperone proteins under both baseline and ER stress conditions. Therefore, pools derived from standard CHO and MMP-enriched hosts were evaluated for expression of PERK and ER chaperone proteins, BiP and PDI, using Western blot analysis. Reduced levels of PERK, BiP and PDI were observed in MMP-enriched host-derived pools (Figure 8C), consistent with the inhibitory role of elevated Mfn2 on ER stress observed in other cell types. Finally, mGPDH, an essential component of the mammalian respiratory chain, links mitochondrial and cytosolic processes and plays a key role in maintaining cellular oxidative phosphorylation, lipid metabolism and mitochondrial membrane potential. Thus, upregulation of mGPDH may be another mechanism that allows the physiological capacity of MMP-enriched host cells to be improved and deliver superior protein producing pools and clones.

[0075] Example 5 - Evaluation of the performance of MMP-enriched clones in a bioreactor environment To better understand the improved biomanufacturing capabilities of the MMP host-derived clones compared to standard CHO clones, CDE1a (CHO clone expressing DTE1), CDE2a (CHO clone expressing DTE2), MDE1a (MMP-enriched clone expressing DTE1) and MDE2a (MMP-enriched clone expressing DTE2) clones were cultured for 14 days in a 3 L bioreactor under fed-batch conditions representative of a large-scale manufacturing process. The data demonstrated that the MMP host-derived clones MDE1a and MDE2a had a 1.6-fold (2.5 vs. 4.1 g / L, p<0.05) and 1.4-fold (2.7 vs. 3.8 g / L, p<0.04) increase in titer, respectively, compared to the corresponding CHO clones (Figure 12A-B). Similarly, a significant increase in specific productivity was observed for clones generated from the MMP-enriched host (8.1 pg / cell / day of CDE1a vs. 15.2 pg / cell / day of MDE1a, p<0.04, and 9.2 pg / cell / day of CDE2a vs. 14.1 pg / cell / day of MDE2a, p<0.05) (Figure 12C-D). Cell growth patterns (Figure 12E-F), viability (Figure 12G-H) and glucose consumption (Figure 12I-J) remained similar between CHO and MMP clones, but a more favorable lactate profile was observed for both MMP clones over the course of the bioreactor run (Figure 12K-L). Standard CHO clones exhibited higher lactate production with the end of the run lactate spike reflecting a decrease in mitochondrial oxidative capacity associated with reduced recombinant protein production in these clones (Figure 12K-L). Taken together, the results suggest that the MMP-enriched host is better suited to address the challenges of DTE biotherapeutics developability and manufacturing. Furthermore, given these significant improvements in cell performance, this novel host may serve as a cost-effective and high-throughput tool for the cell line development process to produce recombinant proteins in various molecular formats.

Claims

1. A method for improving the cell cloning efficiency of recombinant host cells for expressing a polypeptide of interest, comprising isolating host cells having a high level of mitochondrial membrane potential (MMP), transfecting the host cells with an expression plasmid encoding the polypeptide of interest, and growing the cells under conditions that promote cell growth and production of the polypeptide.

2. At least 90% of the cells have a cell population >10 as determined by flow cytometry. ３ The method of claim 1, having a log MMP fluorescent staining intensity.

3. The method of claim 2, wherein the dye used in the flow cytometry analysis is Mito-ID, Rh123, DioC6, JC-1, or tetramethylrhodamine methyl ester (TMRM).

4. The method of any one of claims 1 to 3, wherein the level of the MMP is determined by flow cytometry.

5. 2. The method of claim 1, wherein the recombinant cell is a Chinese hamster ovary (CHO) cell.

6. 10. The method of claim 1, wherein the recombinant cells are contained in a cell culture, and the cell culture is a batch culture, a fed-batch culture, a continuous culture, or a perfusion culture.

7. The method of claim 1 , wherein the recombinant cells are adapted to grow in suspension.

8. 8. The method of claim 6 or 7, wherein the cells are cultured in a bioreactor.

9. The method of claim 1 , wherein the recombinant cell stably expresses the polypeptide of interest.

10. 10. The method of claim 9, wherein the polypeptide of interest is produced at a level of at least 10 pg / cell / day, at least 15 pg / cell / day, at least 20 pg / cell / day, or at least 25 pg / cell / day.

11. 10. The method of claim 1, wherein the recombinant cell has undergone at least 25, at least 50, at least 75, or at least 100 divisions.

12. The method of claim 6, wherein the viability of the cells is increased compared to a parental CHO cell culture in which at least 90% of the cells have an MMP fluorescent staining intensity of <10 3 log as determined by flow cytometry.

13. 10. The method of claim 1, wherein the recombinant cell has increased levels of mGPDH, GAS7, and Mfn2 gene expression or has been engineered to overexpress mGPDH, GAS7, and / or Mfn2.

14. A recombinant host cell for use in the method of claim 1, wherein the host cell has a stably high level of mitochondrial membrane potential (MMP), and at least 90% of the cells in a culture of the host cell have an MMP fluorescent staining intensity of >103 log as determined by flow cytometry.

15. The recombinant host cell described in claim 14, which is a Chinese hamster ovary (CHO) cell.

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