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ABSTRACT Chinese hamster ovary (CHO) cells, the major mammalian host cells for biomanufacturing of therapeutic proteins, have been extensively investigated to enhance productivity and product quality. However, cell line instability resulting in unexpected changes in productivity or product quality continues to be a challenge. Based on previous reports about causes and characteristics of production instability, we hypothesized that chromosomal rearrangements due to genomic instability are associated with production instability and that these events can be characterized. We developed a production instability model using secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) as well as a framework to quantify chromosomal rearrangements by karyotyping. In the absence of methotrexate (MTX), CHO-SEAP cells exhibited a slightly increased growth rate, a significantly decreased specific productivity, and changes in the chromosomal rearrangement ratio of seven chromosomes. In contrast, when MTX was re-introduced, the growth rate and SEAP productivity reversed to the initial values, demonstrating the reversibility of production instability in CHO-SEAP cells. Fluorescence in situ hybridization analysis identified that the SEAP genes were incorporated in the chromosomal rearrangement (insertion) part of the der(Z9) chromosome. Karyotype analysis indicated that the insertion ratio of the der(Z9) chromosome decreased in the CHO-SEAP cells grown without MTX, demonstrating a correlation between chromosomal rearrangement and production instability. Our results support a mechanism for production instability, wherein a randomly generated chromosomal rearrangement (or genotype) results in cells with a growth advantage that is also associated with non (or low)-producing traits. As a result, the non-producing cells grow faster and thereby outgrow the producing population. Biotechnol. Bioeng. 2017;114: 1045-1053. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

To understand the intracellular responses in recombinant Chinese hamster ovary (rCHO) cells adapted to grow in serum-free suspension culture, a proteomic approach was employed. After rCHO cells producing erythropoietin were adapted to grow in suspension culture with the two different serum-free media (SFM4CHO™ and SF-L1), proteome analyses were carried out using 2-D PAGE and based on spot intensities, 58 high-intensity protein spots were selected. Of the 58 protein spots, which represented 34 different kinds of proteins, 55 were identified by MALDI-TOF-MS, and MS/MS. Compared with the results in serum-containing medium, six proteins, four de novo synthesis of nucleotides-related proteins (dihydrolipoamide S-acetyltransferase, transaldolase, inosine-5′-monophosphate dehydrogenase 2, and lymphoid-restricted membrane protein) and two molecular chaperones (heat shock protein 70 kDa and 60 kDa [HSC70, HSP60]) were significantly increased in SFM4CHO™. From the results of proteomic analysis, HSP60 and HSC70, which were increased in both SFM, were selected as candidate proteins for engineering and rCHO cell lines overexpressing these genes were constructed. Cells overexpressing HSP60 and/or HSC70 showed 10-15% enhanced cell concentration during serum-free adaptation and 15-33% reduction in adaptation time. Taken together, identification of differentially expressed proteins in rCHO cells by a proteomic study can provide insights into understanding the intracellular events and clues to find candidate genes for cell engineering for improved performance of rCHO cells during adaptation to serum-free suspension culture. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011 [ABSTRACT FROM AUTHOR]

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull-down assay was performed with dual-tagged (N-terminal GST- and C-terminal hexahistidine-tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual-tagged EPO were then resolved by two-dimensional gel electrophoresis (2DE) and identified by MALDI-TOF MS/MS. A total of 27 protein spots including glucose-regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull-down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [ABSTRACT FROM AUTHOR]