Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture

Abstract

Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28-34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system.

Fig 1. Kinetics of cell growth and rh-tPA production.

Recombinant CHO TF70R viable cell density (A), percentage of cell viability (B), rh-tPA production (C), cell-specific rh-tPA production (D), glucose consumption (E), lactate production (F), and glutamine consumption (G) of cell cultures at 37°C (closed squares), and under temperature downshift (TDS) to 30°C at 48 h (open circles). Dotted line denotes TDS from 37 to 30°C, and transcriptomic samples were taken at 24 and 48 h after TDS.

Fig 2. Transcript abundance analysis of 21,789 genes after temperature downshift (TDS).

Differential expression determined by Illumina sequencing of duplicated samples collected at 24 h (A) and 48 h (B) after TDS of culture and compared to the control culture at 37°C, sampled at 48 h of culture (bioinformatic analysis in Materials and Methods). Coding DNA sequence data in each condition were analyzed with the non-parametric and data-adaptive algorithm NOISeq R package [29], following TMM normalization. Genes without differential expression are represented by light gray dots. Genes with differential expression (>1.6 fold change) are shown in dark gray with q of 0.6–0.8 (A), and q of 0.9–0.95 (B). Those that are differentially expressed (>1.6 fold change) with q > 0.8 (A) and q > 0.95 (B) are shown in black dots.

Fig 3. Enriched gene ontology annotation of differentially expressed genes.

In total, 171 genes at 24 h after TDS (>1.6 fold change, q > 0.8) and 995 genes at 48 h after TDS (>1.6 fold change, q > 0.95) were classified in sub-ontologies. Enrichment scores on the y-axis and x-axis bar charts represent the follows: GO type—A, Biological process; B, Cellular component; and C, Molecular function. (CPM) cellular protein modulation, (SGTP ST) small GTPase signal transduction, (MBM) microtubule-based movement, (R GTP ST) regulation of GTPase signal transduction, (APP) antigen process and presentation, (R RGT PA) regulation of Rab GTPase activity, (CSD) chromatin assembly or disassembly, (CD PS) cyclin-dependent protein serin.

Fig 4. Pictorial representation of transcripts expressed differentially in response to moderate hypothermia after 24 or 48 h of exposition.

Differentially expressed genes after 24 h of TDS, up regulated genes are presented in orange; down regulated genes are presented in blue. Differentially expressed genes after 48 h of TDS, up regulated genes are presented in red; down regulated genes are presented in green. Black arrows and red lines represent stimulation and inhibition, respectively.