Microarray and proteomics expression profiling identifies several candidates, including the valosin‐containing protein (VCP), involved in regulating high cellular growth rate in production CHO cell lines

Abstract

A high rate of cell growth (micro) leading to rapid accumulation of viable biomass is a desirable phenotype during scale up operations and the early stages of production cultures. In order to identify genes and proteins that contribute to higher growth rates in Chinese hamster ovary (CHO) cells, a combined approach using microarray and proteomic expression profiling analysis was carried out on two matched pairs of CHO production cell lines that displayed either fast or slow growth rates. Statistical analysis of the microarray and proteomic data separately resulted in the identification of 118 gene transcripts and 58 proteins that were differentially expressed between the fast- and slow-growing cells. Overlap comparison of both datasets identified a priority list of 21 candidates associated with a high growth rate phenotype in CHO. Functional analysis (by siRNA) of five of these candidates identified the valosin-containing protein (VCP) as having a substantial impact on CHO cell growth and viability. Knockdown of HSPB1 and ENO1 also had an effect on cell growth (negative and positive, respectively). Further functional validation in CHO using both gene knockdown (siRNA) and overexpression (cDNA) confirmed that altered VCP expression impacted CHO cell proliferation, indicating that VCP and other genes and proteins identified here may play an important role in the regulation of CHO cell growth during log phase culture and are potential candidates for CHO cell line engineering strategies.

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

“…In the case of the proteasome, we only find Psmd3 (Rpn3), a structural component of the lid of the 26S proteasome, significantly changing (q<0.05) with increasing qP. Such a general trend towards down-regulation of the structural genes of the proteasome [50,60,61] or related ubiquitin ligases [7] has also been found in previous studies, and was also identified in recombinant yeast [62]. In combination with the results for the ER it seems that, although the ER is upregulated for high protein cargo, in the high producing subclones this is not happening under stress response.…”

Mammalian cells as biopharmaceutical production hosts in the age of omics

“…Five of these genes were selected as engineering targets to analyze their effect on cell densities for mAb-producing CHO K1 cells: valosincontaining protein (VCP), enolase 1 (ENO), heat shock 27-kDa protein 1 (HSPB1), BiP and actin-related protein (ACTR1A). VCP, ENO, HSPB1, and BiP were up-regulated at the protein level, while ACTR1A was down-regulated [12].…”

“…To identify relevant targets for cellular engineering, Doolan et al [12] used transcriptomics and proteomics to compare two pairs of CHO cell lines with different growth rates: two antibody-producing CHO K1 cell lines with growth rates of 0.014 and 0.035/h; and two untransfected CHO DUKX cell lines with growth rates of 0.024 and 0.042/h. Using custom-made WyeHamster2a oligo microarrays, the authors identified 118 differentially expressed genes (fold change (FC) ≥ 1.2) common to both pairs of fast-and slow-growing cells [12]. Proteomic analysis (2D difference gel electrophoresis (2D DIGE), matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)-MS and LC-MS/MS) revealed 34 differentially regulated protein spots (FC ≥ 1.5) in the antibody-producing CHO K1 pair, and 24 in the untransfected CHO DUKX pair.…”

“…Consequently, several omics studies have investigated cellular characteristics affecting cell densities. Examples include the metabolic shift [10], apoptosis [11], and growth rate [12].…”

Development and characterization of a Chinese hamster ovary cell-specific oligonucleotide microarray

“…The microarray has been used to study the effects of overexpression of soluble PACE on CHO cells expressing a transgene for bone morphogenic protein-2 (Doolan et al 2008), predicting productivity in CHO (Clarke et al 2011), as well as to identify genes linked with the ability of CHO cells to modulate their rate of growth (Doolan et al 2010). …”