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.

Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody

“…We anticipated that some culture conditions could potentially alter impurity formation. In particular, culture temperature affects transcript levels in CHO cells (Bedoya‐Lopez et al, ; Gomez et al, ; Mason, Sweeney, Cain, Stephens, & Sharfstein, ; Oguchi, Saito, Tsukahara, & Tsumura, ; Yoon, Kim, & Lee, ) that could potentially mediate B‐Ab folding or correct chain assembly. We found culture temperature modulated two product‐related impurities in cell line X: half B‐Ab and high‐molecular‐weight (HMW) species.…”

Cell-culture growth conditions resulting in the oxidation of a recombinant antigen-binding fragment

“…Upon shifting the temperature at day 6, no significant difference in glucose utilization among the bioreactors was observed, and some of the bioreactors demonstrated low to no lactate accumulation. The low lactate accumulation may be due to the cells mimicking the stationary phase of the cell cycle as supported by recent studies, which have reported lactate consumption during the stationary phase in fed batch cultures (Bedoya-López et al 2016;Trummer et al 2006;Furukawa and Ohsuye 1998;Young 2013;Mulukutla et al 2012;Cruza et al 2000;Lao and Toth 1997).…”

Sub physiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells

“…In this manuscript, we describe the utilisation of RNASeq to characterise transcriptomics changes induced by temperature shift of a mAb producing CHOK1 cell line. For the initial phase of our analysis we sought, similar to other studies (Bedoya-López et al, 2016;Tossolini, López-Díaz, Kratje, & Prieto, 2018), to identify gene-level changes in expression following a decrease in cell culture temperature. Using a count-based differential expression method the abundance of > 1,300 genes were found to be significantly altered 24hrs after the temperature was reduced from 37°C to 31°C.…”