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This study explored the cell line-specific effects of dexamethasone (DEX) on three antibody-producing Chinese hamster ovary (CHO) clones derived from different parental host cell lines: CHO-K1, -S, and -DG44. The CHO-K1 and -DG44 cell lines displayed DEX-concentration-dependent growth inhibition but had improved viability in later culture stages, extending culture longevity. Conversely, the CHO-S cell line was less sensitive to DEX regarding cell growth but exhibited a drastic decrease in viability in the later stages of culture. The cell line-specific impact of DEX (i.e. the anti-apoptotic or pro-apoptotic effects) could have resulted from the presence or absence of the GRα-D isoform and relative level of GRα-C isoform in these cell lines. The CHO-K1 and -DG44 cell lines, which expressed GRα-D and relatively low levels of GRα-C, displayed improved cell viability in response to DEX. In contrast, the CHO-S cell line, which lacked GRα-D expression with relatively higher levels of GRα-C, exhibited pro-apoptotic effects upon exposure to DEX. DEX also influenced glucose consumption and metabolite production in all three cell lines, while its effects on antibody quality were negligible. Therefore, when supplementing glucocorticoids such as DEX in cell culture media, it is crucial to assess their ability to enhance culture performance carefully. This study could provide insights for optimizing production media to specific cell lines within the bioprocessing field. [ABSTRACT FROM AUTHOR]

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. Key points: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing [ABSTRACT FROM AUTHOR]

Identification of recombinant gene integrations sites in the Chinese hamster ovary (CHO) cell genome is increasingly important to assure monoclonality. While next-generation sequencing (NGS) is commonly used for the gene integration site analysis, it is a time-consuming and costly technique as it analyzes the entire genome. Hence, simple, easy, and inexpensive methods to analyze transgene insertion sites are necessary. To selectively capture the integration site of transgene in the CHO genome, we applied splinkerette-PCR (spPCR). SpPCR is an adaptor ligation-based method using splinkerette adaptors that have a stable hairpin loop. Restriction enzymes with high frequencies in the CHO genome were chosen using a Python script and used for the in vitro spPCR assay development. After testing on two CHO housekeeping genes with known loci, the spPCR-based genome walking technique was successfully applied to recombinant CHO cells to identify the transgene integration site. Finally, the comparison with NGS methods exhibited that the time and cost required for the analysis can be substantially reduced. Taken together, the established technique would aid the stable cell line development process by providing a rapid and cost-effective method for transgene integration site analysis. • Identification of transgene location is critical to assure monoclonality of CHO cells. • spPCR-based genome walking assay was developed and applied to recombinant CHO cells. • Python script-assisted selection of restriction enzyme can improve spPCR success rate. • spPCR saves required analysis time and cost substantially compared to current methods. • spPCR may aid cell line development by providing a simple and rapid analysis platform. [ABSTRACT FROM AUTHOR]

Methotrexate (MTX)-mediated gene amplification has been widely used in Chinese hamster ovary (CHO) cells for the biomanufacturing of therapeutic proteins. Although many studies have reported chromosomal instability and extensive chromosomal rearrangements in MTX-mediated gene-amplified cells, which may be associated with cell line instability issues, the mechanisms of chromosomal rearrangement formation remain poorly understood. We tested the impact of DNA double-strand breaks (DSBs) on chromosomal rearrangements using bleomycin, a DSB-inducing reagent. Bleomycin-treated CHO-DUK cells, which are one of the host cell lines deficient in dihydrofolate reductase (Dhfr) activity, exhibited a substantial number of cells containing radial formations or non-radial formations with chromosomal rearrangements, suggesting that DSBs may be associated with chromosomal rearrangements. To confirm the causes of DSBs during gene amplification, we tested the effects of MTX treatment and the removal of nucleotide base precursors on DSB formation in Dhfr-deficient (i.e., CHO-DUK) and Dhfr-expressing (i.e., CHO-K1) cells. Immunocytochemistry demonstrated that MTX treatment did not induce DSBs per se, but a nucleotide shortage caused by the MTX-mediated inhibition of Dhfr activity resulted in DSBs. Our data suggest that a nucleotide shortage caused by MTX-mediated Dhfr inhibition in production cell lines is the primary cause of a marked increase in DSBs, resulting in extensive chromosomal rearrangements after gene amplification processes. [ABSTRACT FROM AUTHOR]