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

Abstract

Heterogeneity in gene expression and phenotypic traits is an inherent feature within the "clonal" cell lines used for biopharmaceutical production. This feature can allow the selection of cell lines with improved phenotypes and adaptation to growth under preferential conditions to improve productivity or provide a platform to study the molecular basis of improved characteristics. A repeated process of extended batch culture of a recombinant antibody producing GS-NS0 myeloma cell line generated a stable variant cell line displaying increased resistance to both environmental stresses and chemical apoptosis inducers, and resulted in extended culture viability and increased antibody production. An interesting feature of the variant cell line was an altered metabolic state with consumption of lactate as the culture progressed. The variant cell line also showed altered expression of proteins associated with autophagy suggesting a role for this process in extending cell survival in culture. Targeted transcriptomic analysis was carried out on the parental and variant cell lines using a qRT-PCR array containing a panel of apoptosis-associated genes to elucidate both the predominant apoptotic pathways in the NS0 cell line with batch culture progression, and the altered gene expression contributing to increased survival in the variant line. Results indicated a balance between pro- and anti-apoptotic signaling is triggered with the onset of cell death in the NS0 cell line. Pro-survival pathways such as NFκB signaling and the unfolded protein response were implicated along with death receptor, endoplasmic reticulum stress and p53 mediated apoptotic pathways. The identification of altered gene expression in the variant cell line also provides several potential targets for cell engineering strategies to create improved cell lines for production.

Cell cycle and apoptosis: a map for the GS-NS0 cell line at the genetic level and the link to environmental stress

“…The batch cultures started with a high fraction of cells in the G 1 /G 0 phase, which rapidly left this state in order to join the proliferating population. Soon after, glutamate deprivation occurred at around 50 h of culture, whereby atf5 upregulation peaked (50% higher) suggesting that glutamate deprivation is among the first factors that introduce metabolic stress, in agreement with previous results [5]. The upregulation of atf5 triggered the upregulation of bcl-2 (which followed at around 90 h).…”

The relationship between mTOR signalling pathway and recombinant antibody productivity in CHO cell lines

“…However, the mechanism underlying these improvements has not yet been elucidated. We believe that using a transcriptomic approach, such as the pathway-focused polymerase chain reaction (PCR) array, which has proven to be useful in a similar study [47], we can elucidate the connections between mTOR, cell growth, and productivity. Thus, this study initially employed two recombinant CHO cell lines with a 17.4-fold difference in specific productivity to study the differential expressions of genes related to the mTOR signaling pathway.…”

Autophagy induced by rapamycin and carbon‐starvation have distinct proteome profiles in Aspergillus nidulans

“…Such regulation is repressed and autophagy is induced in the presence of nutrient starvation or the macrolide antibiotic rapamycin (sirolimus, Abeliovich, 2004). Recent studies have also shown that autophagy plays an important survival role in higher eukaryotes (Browne and Al-Rubeai, 2011;Kim et al, 2009) and it has been suggested to be important in developing improved eukaryotic strains for biotechnology applications (Zustiak et al, 2008).…”