Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium.

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine-the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfection of Gs-Enbrel and Aspg-Enbrel plasmids.

Factors affecting the quality of therapeutic proteins in recombinant Chinese hamster ovary cell culture.

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the commercial production of therapeutic proteins. Fed-batch culture is widely used to produce therapeutic proteins, including monoclonal antibodies, because of its operational simplicity and high product titer. Despite technical advances in the development of culture media and cell cultures, it is still challenging to maintain high productivity in fed-batch cultures while also ensuring good product quality.

Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures.

Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells.

Reprogramming AA catabolism in CHO cells with CRISPR/Cas9 genome editing improves cell growth and reduces byproduct secretion.

Chinese hamster ovary (CHO) cells are the preferred host for producing biopharmaceuticals. Amino acids are biologically important precursors for CHO metabolism; they serve as building blocks for proteogenesis, including synthesis of biomass and recombinant proteins, and are utilized for growth and cellular maintenance. In this work, we studied the physiological impact of disrupting a range of amino acid catabolic pathways in CHO cells.

BiP Inducer X: An ER Stress Inhibitor for Enhancing Recombinant Antibody Production in CHO Cell Culture.

Prolonged endoplasmic reticulum (ER) stress reduces protein synthesis and induces apoptosis in mammalian cells. When dimethyl sulfoxide (DMSO), a specific monoclonal antibody productivity (q )-enhancing reagent, is added to recombinant Chinese hamster ovary (rCHO) cell cultures (GSR cell line), it induces ER stress and apoptosis in a dose-dependent manner. To determine an effective ER stress inhibitor, three ER stress inhibitors (BiP inducer X [BIX], tauroursodeoxycholic acid, and carbazole) are examined and BIX shows the best production performance.

Revealing Key Determinants of Clonal Variation in Transgene Expression in Recombinant CHO Cells Using Targeted Genome Editing.

Generation of recombinant Chinese hamster ovary (rCHO) cell lines is critical for the production of therapeutic proteins. However, the high degree of phenotypic heterogeneity among generated clones, referred to as clonal variation, makes the rCHO cell line development process inefficient and unpredictable. Here, we investigated the major genomic causes of clonal variation.

Baicalein Reduces Oxidative Stress in CHO Cell Cultures and Improves Recombinant Antibody Productivity.

Oxidative stress that naturally accumulates in the endoplasmic reticulum (ER) as a result of mitochondrial energy metabolism and protein synthesis can disturb the ER function. Because ER have a responsibility on the protein synthesis and quality control of the secreted proteins, ER homeostasis has to be well maintained. When H O , an oxidative stress inducer, is added to recombinant Chinese hamster ovary (rCHO) cell cultures, it reduced cell growth, monoclonal antibody (mAb) production, and galactosylated form of mAb in a dose-dependent manner.

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion.

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation.

Understanding of altered N-glycosylation-related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting.

To understand the effects of ammonium on N-glycosylation, recombinant Chinese hamster ovary (rCHO) cells that produce the Fc-fusion protein were cultivated in serum-free suspension cultures with 10 mM ammonium addition. The addition of ammonium to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of an Fc-fusion protein. Fifty two N-glycosylation-related gene expressions were assessed by the NanoString nCounter system, which provides a digital readout using custom-designed color-coded probes.

Effect of lithium chloride on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture.

Lithium chloride (LiCl), which is a specific inhibitor of glycogen synthase kinase-3β, is known to induce cell cycle arrest at the G2/M phase and to regulate apoptosis. To determine the potential of LiCl as a chemical additive to enhance specific productivity (q p) of recombinant Chinese hamster ovary (rCHO) cells through cell cycle arrest at G2/M phase, rCHO cells producing Fc-fusion protein were cultivated in serum-free media with LiCl concentrations ranging from 0 to 20 mM. The addition of LiCl induced cell cycle arrest at G2/M phase and thereby decreased the specific cell growth rate.

Effect of glutamine substitution by TCA cycle intermediates on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture.

In an effort to reduce the accumulation of ammonia in culture medium, three different TCA cycle intermediates, (alpha-ketoglutarate (α-KG), citric acid and succinic acid) along with glutamic acid for a comparison, were examined as a substitute for glutamine with rCHO cell line producing a Fc-fusion glycoprotein. Among them, α-KG showed the best production performance. When cells were cultivated with 4 mM α-KG, the final ammonia concentration did not exceed 3 mM, which is less than one fourth of that with 4 mM glutamine.

Effect of Bcl-x(L) overexpression on lactate metabolism in chinese hamster ovary cells producing antibody.

Previously, overexpression of anti-apoptotic proteins, such as E1B-19K and Aven, was reported to alter lactate metabolism of CHO cells in culture. To investigate the effect of Bcl-xL , a well-known anti-apoptotic protein, on lactate metabolism of recombinant CHO (rCHO) cells, two antibody-producing rCHO cell lines with regulated Bcl-xL overexpression (CS13*-0.02-off-Bcl-xL and CS13*-1.

Current state and perspectives on erythropoietin production.

Erythropoietin is a major regulator of erythropoiesis which maintains the body's red blood cell mass and tissue oxygenation at an optimum level. Recombinant human erythropoietin (rhEPO), which is a widely used therapeutic agent for the treatment of anemia and which represents one of the largest biopharmaceuticals markets, is produced from recombinant Chinese hamster ovary cells. rhEPO is a glycoprotein with complex glycan structure, which is responsible for its therapeutic efficacy, including the in vivo activity and half-life.

Bcl-x(L) overexpression delays the onset of autophagy and apoptosis in hyperosmotic recombinant Chinese hamster ovary cell cultures.

Hyperosmolality in recombinant Chinese hamster ovary (rCHO) cell cultures induces autophagy and apoptosis. To investigate the effect of Bcl-x(L) overexpression on autophagy and apoptosis in hyperosmotic rCHO cell cultures, an erythropoietin (EPO)-producing rCHO cell line with regulated Bcl-x(L) overexpression was subjected to hyperosmolality resulting from NaCl addition in a batch culture and nutrient supplementation in a fed-batch culture. In the batch culture, Bcl-x(L) overexpression suppressed apoptosis, as evidenced by a decreased amount of cleaved caspase-7 and PARP.

Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed-batch culture: effect of nutrient supplementation.

Upon nutrient depletion during recombinant Chinese hamster ovary (rCHO) cell batch culture, cells are subjected to apoptosis, type I programmed cell death (PCD), and autophagy which can be type II PCD or a cell survival mechanism. To investigate the effect of nutrient supplementation on the two PCDs and protein production in rCHO cells, an antibody-producing rCHO cell line was cultivated in batch and fed-batch modes. The feed medium containing glucose, amino acids, and vitamins was determined through flask culture tests and used in bioreactor cultures.