Transcriptional Profiling of Gene Expression Changes in a PACE-Transfected CHO DUKX Cell Line Secreting High Levels of rhBMP-2

Abstract

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry for the production of recombinant human proteins including complex polypeptides such as recombinant human bone morphogenic protein 2 (rhBMP-2). Large-scale manufacture of rhBMP-2 has associated production difficulties resulting from incomplete processing of the recombinant human protein due to insufficient endogenous levels of the paired basic amino acid cleaving enzyme (PACE) in CHO. In order to resolve this issue, CHO DUKX cells expressing rhBMP-2 were transfected with the soluble version of human PACE (PACEsol) resulting in improved amino-terminal homogeneity and a fourfold increase in rhBMP-2 productivity. In this article, we present a microarray expression profile analysis comparing the parental lineage to the higher producing subclone co-expressing PACEsol using a proprietary CHO-specific microarray. Using this technology we observed 1,076 significantly different genes in the high-productivity cells co-expressing PACEsol. Following further analysis of the differentially expressed genes, the Unfolded Protein Response (UPR) component of the endoplasmic reticulum stress response pathway was identified as a key candidate for effecting increased productivity in this cell system. Several additional ER- and Golgi-localised proteins were identified which may also contribute to this effect. The results presented here support the use of large-scale microarray expression profiling as a viable and valuable route towards understanding the behaviour of bioprocess cultures in vitro.

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…Indeed, as recently stated by Clarke and Lee (2014), the CHO community is currently generating 'omics data at an ever-increasing rate. This is supported by the following non-exhaustive examples on CHO 'omics studies: genomics (Lewis et al, 2013;Xu et al, 2011), transcriptomics (Becker et al, 2011;Doolan et al, 2008), translatomics (Courtes et al, 2013), proteomics (BaycinHizal et al, 2012;Carlage et al, 2009), metabolomics (Selvarasu et al, 2012;Zang et al, 2011) and integrative 'omics (Clarke et al, 2012). Moreover, a consensus genome-scale reconstruction of…”

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

“…In fact, Clarke et al 2011 found a set of 287 genes for the prediction of cellular productivity in the model they established. However, as this study was based on an early microarray chip that contained only ~3500 expressed CHO genes out of the estimated 15 000 transcripts present in a cell at any time [9], it is likely that many more mRNAs and other factors like non-coding RNAs (ncRNAs) play a role in determining productivity [10].…”

Dynamics of unfolded protein response in recombinant CHO cells

“…A comprehensive study of these genes in recombinant CHO cells is essential for a better understanding of the pathway and its response. Majority of existing studies are limited to a few chosen genes from these pathways and to few time-points (Murphy et al 2001;Underhill et al 2005;Cudna and Dickson 2006;Doolan et al 2008;Ku et al 2008). In this work, we undertake dynamic profiling of all major ER chaperones and UPR pathway genes in a batch culture.…”