Mild hypothermia upregulates <i>myc</i> and <i>xbp1s</i> expression and improves anti-TNFα production in CHO cells

<div><p>Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on <i>myc</i> and <i>xbp1s</i> expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant <i>anti-TNFα</i> and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of <i>anti-TNFα</i>. Under mild hypothermia <i>myc</i> and <i>xbp1s</i> expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of <i>myc</i> and <i>xbp1s</i> in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.</p></div

Relationship between q_anti-TNFα and q (lactate: ●, and glutamine: ●; both after shift).

<p>Relationship between q_anti-TNFα and q (lactate: ●, and glutamine: ●; both after shift).</p

Differential expressions of mRNA encoding for anti-TNFα, Myc and XBP1s in CN1 and CN2 at 37°C (■), 33°C (■) and 31°C (■).

<p>The relative expressions of <i>anti-TNFα</i>, <i>myc</i> and <i>xbp1s</i> were analysed at 6 and 72 h of culture using RT-qPCR and <i>gapdh</i> as reference gene. The results are expressed and plotted in log₁₀. Significant differences (α = 0.05) between temperature and clones according to the Tukey HSD test are indicated by the p-values in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194510#pone.0194510.s007" target="_blank">S5 Table</a> (Supplementary file). Differences in the expression levels of <i>anti-TNFα</i>, <i>xbp1s</i> and <i>myc</i> between both culture periods (i.e. 6 h vs 72 h of culture) were evaluated by a <i>t</i>-test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194510#pone.0194510.s006" target="_blank">S4 Table</a>, Supplementary file) and expressed in the figure as: * p < 0.05, ** p < 0.01 and p < 0.001.</p

Physiological and metabolic parameters of CN1 and CN2 growing at 37, 33 and 31°C.

<p>Physiological and metabolic parameters of CN1 and CN2 growing at 37, 33 and 31°C.</p

Principal component analysis (PCA) using 11 physiological parameters of CN1 and CN2 at 37, 33 and 31°C.

<p>The variables analysed were maximum cell density (X_max), specific cell growth rate (<i>μ</i>), yield of lactate from glucose (Y_lac/glc), specific anti-TNFα productivity (q_anti-TNFα), maximum anti-TNFα production (anti-TNFα_max), volumetric productivity (Q_vol), specific consumption rate of glucose (q_glc), glutamine (q_gln), and lactate after shift (q_lac2); specific production rate of lactate (q_lac) and glutamine after shift (q_gln2). A: correlation circle showing relation between the measured parameters; B: Cluster analysis (Ward’s method) of the observations using Euclidian distance; C and D: Ordination of the first and second principal component (PC1 and PC2, respectively), showing observation distribution. Different conditions are plotted using circles to identify the effect of clones (C) and temperature (D).</p