Transcriptomic responses to sodium chloride‐induced osmotic stress: A study of industrial fed‐batch CHO cell cultures

Abstract

The rapidly expanding market for monoclonal antibody and Fc-fusion-protein therapeutics has increased interest in improving the productivity of mammalian cell lines, both to alleviate capacity limitations and control the cost of goods. In this study, we evaluated the responses of an industrial CHO cell line producing an Fc-fusion-protein to hyperosmotic stress, a well-known productivity enhancer, and compared them with our previous studies of murine hybridomas (Shen and Sharfstein, Biotechnol Bioeng. 2006;93:132-145). In batch culture studies, cells showed substantially increased specific productivity in response to increased osmolarity as well as significant metabolic changes. However, the final titer showed no substantial increase due to the decrease in viable cell density. In fed batch cultures, hyperosmolarity slightly repressed the cellular growth rate, but no significant change in productivity or final titer was detected. To understand the transcriptional responses to increased osmolarity and relate changes in gene expression to increased productivity and repressed growth, proprietary CHO microarrays were used to monitor the transcription profile changes in response to osmotic stress. A set of osmotically regulated genes was generated and classified by extracting their annotations and functionalities from online databases. The gene list was compared with results previously obtained from similar studies of murine-hybridoma cells. The overall transcriptomic responses of the two cell lines were rather different, although many functional groups were commonly perturbed between them. Building on this study, we anticipate that further analysis will establish connections between productivity and the expression of specific gene(s), thus allowing rational engineering of mammalian cells for higher recombinant-protein productivity.

Fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity

“…cDNAs were prepared from mRNAs extracted from YB2/0 cells expressing IgG1 B that were sampled on days 6, 9, 11, and 12 during serum-free 1-L fed-batch culture in hypoosmotic (250 mOsm/kg) and hyperosmotic (410 mOsm/kg) media. Microarray analyses indicated that hypoosmotic conditions led to the downregulation of glycolysis gene expression, particularly pyruvate kinase and aldolase (data not shown), which is in agreement with the proteomic and transcriptomic analyses of the effects of osmotic stress on CHO and hybridoma (Shen and Sharfstein 2006;Shen et al 2010). Next, we used semi-quantitative RT-PCR to evaluate gene expression under hypoosmotic versus hyperosmotic conditions and observed three marked responses (Table 3).…”

Analysis of an artificially selected GS‐NS0 variant with increased resistance to apoptosis

“…Mitochondrial signaling was also heavily implicated in this study although there was no evidence of ER stress mediated apoptosis (Wong et al, 2006). Discrepancies in the stress responses between hamster and murine cell lines have also been seen in other studies in spite of a high level of sequence similarity (Shen et al, 2010) highlighting the importance of having a fuller understanding of the signaling mechanisms in a particular line, or for a particular set of conditions before embarking on process optimization and cell engineering experiments.…”

Chemostat‐based transcriptional analysis of growth rate change and BCL‐2 over‐expression in NS0 cells

“…Increasingly, efforts have been made to probe transcriptomic and proteomic alterations between cells of different productivities (Charaniya et al, 2009;Khoo et al, 2007;Seth et al, 2007;Smales et al, 2004), culture conditions (Krampe et al, 2008;Shen et al, 2010;Swiderek and Al-Rubeai, 2007) or cells treated with chemical agents (Kantardjieff et al, 2010). Indeed, ''omic'' data available for mammalian cells have so far mainly been obtained in conventional batch/fed-batch shaker or bioreactor cultures.…”