Principal Component Analysis (PCA).

<p>A, B and C: Hyperplane between the first and second principal components (PC1 and PC2, respectively). A, B and C show the same hyperplane, but the symbols are coloured differently in each inset to indicate the factors studied. D: Dendrogram (Ward distance) of the observation used in PCA to identify the two defined groups in A, B and C. E: Loadings of the first principal component (PC1) shown in A, B or C (bars represent C.I. 95%).</p

Differential effect of culture temperature and dilution rate on protein production and mRNA expression of rht-PA.

<p>A: Specific productivity of rht-PA. B: Log fold change of mRNA of rht-PA. ▪ Condition 37°C; □ Condition 33°C.</p

Metabolic profile of CHO cells growing at different culture temperatures and dilution rates.

<p>A: Glucose concentration. B: Lactate concentration. C: Glutamate concentration. • Condition 37°C and 0.017 h⁻¹; ○ Condition 33°C and 0.017 h⁻¹; ▴ Condition 37°C and 0.012 h⁻¹; ▵ Condition 33°C and 0.012 h⁻¹. Black dotted line: Start of SS 0.017 h⁻¹. Red dotted line: Start of SS 0.012 h⁻¹.</p

Profile of CHO cells growing, viability and rht-PA production at different culture temperatures and dilution rates.

<p>A: Viable cell concentration. B: Viability percentage. C: rht-PA concentration. • Condition 37°C and 0.017 h⁻¹; ○ Condition 33°C and 0.017 h⁻¹; ▴ Condition 37°C and 0.012 h⁻¹; ▵ Condition 33°C and 0.012 h⁻¹. Black dotted line: Start of SS 0.017 h⁻¹. Red dotted line: Start of SS 0.012 h⁻¹.</p

Intracellular accumulation, specific and volumetric productivity of rht-PA at different culture temperatures and under glycosylation/ERAD inhibition, in batch cultures.

<p>ANOVA and the Tukey’s test. Statistical significance was considered for p<0.05. ^a: Significant differences respect to counterpart culture at different T°; ^b: Significant differences respect to CC; ^c: Significant differences respect to TM; ^d: Significant differences respect to TM/ERAD I.</p

Metabolic behavior of CHO cells growing at different culture temperatures and dilution rates.

<p>A: Specific rate of glucose consumption. B: Specific rate of lactate consumption. C: Specific rate of glutamate consumption. D: Lactate yield from glucose. ▪ Condition 37°C; □ Condition 33°C.</p

CHO cell growth profile in batch culture at different temperatures and under glycosylation/ERAD inhibition.

<p><b>A:</b> Viable cell concentration at 37°C. <b>B:</b> Viable cell concentration at 33°C. ◆ Control culture; ■ Tunicamicyn culture; ▲ Tunicamicyn/ERAD-I culture; ● Tunicamicyn/ERAD-II culture. ◇ Control culture; □ Tunicamicyn culture; △ Tunicamicyn/ERAD-I culture; ○ Tunicamicyn/ERAD-II culture.</p

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

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

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