## 4 Discussion  
The growth, viability, glucose and lactate profiles obtained from the KB26.5-BHRF1 cultures were vastly different than those from the parental cell line. Cell culture expansion was much faster, and the maximum cell density reached 3.92 \(\pm\) 0.03 \(\times\) 10\({}^{6}\) cell/mL, representing a two-fold increase. Additionally, the cell viability profile after the cell density peak remained at over 90% for 24 h after the maximum cell density had been achieved, indicating a delay in apoptosis due to the effects of BHRF1, as anticipated by [PERSON] et al. (2009) [20]. The glucose and lactate profiles showed an initial phase of glucose consumption and lactate generation. The glucose concentration profile decrease was similar to that obtained in the KB26.5 culture, but lactate production rates were reduced by almost half. However, since the cell density almost doubled, the specific glucose consumption rate was affected by BHRF1 expression and, consequently, by lactate production. In order to better quantify the differences, Table 2 presents the main physiological parameters for both cell lines, i.e., growth rate (\(\mu\)), doubling time (t\({}_{\text{d}}\)), specific glucose consumption rate (q\({}_{\text{Glu}}\)), specific lactate production rate (q\({}_{\text{La}}\)), glucose yield on biomass (Y\({}_{\text{Glu}/\text{X}}\)), lactate yield on biomass (Y\({}_{\text{Lac}/\text{X}}\)) and maximum VCD (VCD\({}_{\text{max}}\)). In general terms, the expression of BHRF1 was beneficial for cell physiology and growth. The cell growth rate (\(\mu\)) increased by 78%, the specific glucose consumption rate (q\({}_{\text{Glu}}\)) decreased by 15% and the specific lactate production rate (q\({}_{\text{Lac}}\)) reduced by more than 50%. This resulted in a decrease in both glucose yield on biomass (Y\({}_{\text{Glu}/\text{X}}\)) and lactate yield on biomass (Y\({}_{\text{Lac}/\text{X}}\)) in KB26.5-BHRF1, meaning that more efficient glucose consumption had been achieved, thereby generating less lactate. Overall, this resulted in a more efficient use of the main carbon source. This fact translated into a more than two-fold increase in total cell density under the same media and culture conditions. The results obtained in 2 L bioreactors showed similarities to those observed in shake flask cultures. The maximum cell density was slightly improved for KB26.5, reaching 2.34 \(\pm\) 0.39 \(\times\) 10\({}^{6}\) cell/mL, probably due to the pH control. Even though lactate was produced at similar rates, the lack of pH control in the shake flaks might have negatively affected cell growth, a phenomenon which was reversed in the bioreactor cultures. In the case of KB26.5-BHRF1, the maximum cell density observed was significantly increased, reaching 3.93 \(\pm\) 0.01 \(\times\) 10\({}^{6}\) cell/mL. In addition, the viability strongly decayed after 48 h for KB26.5, while it was maintained at above 90% in BHRF1-KB26.5. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Parameter** & **KB26.5** & **KB26.5-BHRF1** \\ \hline \(\mu\) (h\({}^{-1}\)) & 0.027 \(\pm\) 0.001 & 0.048 \(\pm\) 0.002 \\ t\({}_{\text{d}}\) (h) & 25.500 \(\pm\) 0.928 & 14.560 \(\pm\) 0.627 \\ q\({}_{\text{Glu}}\) (nmol/(mg:h)) & \(-\)802.325 \(\pm\) 71.750 & \(-\)682.100 \(\pm\) 55.536 \\ q\({}_{\text{Lac}}\) (nmol/(mg:h)) & 1337.750 \(\pm\) 175.857 & 663.780 \(\pm\) 13.831 \\ Y\({}_{\text{Glu}/\text{X}}\) (mmol/L)/(10\({}^{6}\) cell/mL) & 14.515 \(\pm\) 0.120 & 4.645 \(\pm\) 0.007 \\ Y\({}_{\text{Lac}/\text{X}}\) (mmol/L)/(10\({}^{6}\) cell/mL) & 24.265 \(\pm\) 1.039 & 5.035 \(\pm\) 1.704 \\ VCD\({}_{\text{max}}\) (10\({}^{6}\) cell/mL) & 1.79 \(\pm\) 0.30 & 3.92 \(\pm\) 0.03 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Main physiological parameters determined for KB26.5 and KB26.5-BHRF1 in shake flask cultures. Values were calculated as the means of data obtained from duplicate cultures.