Dynamic metabolic flux analysis using B-splines to study the effects of temperature shift on CHO cell metabolism

Abstract

a b s t r a c tMetabolic flux analysis (MFA) is widely used to estimate intracellular fluxes. Conventional MFA, however, is limited to continuous cultures and the mid-exponential growth phase of batch cultures. Dynamic MFA (DMFA) has emerged to characterize time-resolved metabolic fluxes for the entire culture period. Here, the linear DMFA approach was extended using B-spline fitting (B-DMFA) to estimate mass balanced fluxes. Smoother fits were achieved using reduced number of knots and parameters. Additionally, computation time was greatly reduced using a new heuristic algorithm for knot placement. B-DMFA revealed that Chinese hamster ovary cells shifted from 37°C to 32°C maintained a constant IgG volumespecific productivity, whereas the productivity for the controls peaked during mid-exponential growth phase and declined afterward. The observed 42% increase in product titer at 32°C was explained by a prolonged cell growth with high cell viability, a larger cell volume and a more stable volume-specific productivity.

Scale-down of CHO cell fed-batch cultures

“…It was also established in this study that the cell size increase is due to a proportional increase in biomass content (Table I), rather than due to a cell swelling effect through the uptake of water and salts. The cell DW measurement resulted in higher values (770 pg/cell or higher) than commonly reported for CHO cells (300-400 pg/cell) 57,85,86 . This can be explained by the larger cell size (17 µ m diameter on day 4) of the CHO cell line used in this study, compared to those reported (12-14 µ m diameter) 57 .…”

“…The cell DW measurement resulted in higher values (770 pg/cell or higher) than commonly reported for CHO cells (300-400 pg/cell) 57,85,86 . This can be explained by the larger cell size (17 µ m diameter on day 4) of the CHO cell line used in this study, compared to those reported (12-14 µ m diameter) 57 . We applied the same DW measurement for another cell line with an average cell diameter of 14 µ m at a viability higher than 90%, which A B resulted in ~330 pg/cell (data not shown).…”

“…Kim et al 58 reported that the enhancement of the specific thrombopoietin productivity is linearly correlated with the increase in cell size for nine CHO cell clones generated using the same transfection process. In a number of studies the relation between cell size and specific productivity was used to enhance mAb productivity, by directly or indirectly increasing the cell size, for example, through cell cycle arrest 53,91 , hyperosmotic pressure 92,93 , and mild hypothermia 57,94 . Acetic acid and formic acid accumulation were observed during the cultures (Figure 2b).…”

“…Furthermore, it was observed that a higher average mAb specific productivity is correlated with conditions that result in a larger cell size (the BC-P clone fed by Actifeed A/B; Table II). Cell specific mAb productivity has been reported to correlate with cell volume 52,56,57 . Kim et al 58 suggested a linear correlation between the increase in specific thrombopoietin productivity and the increase of cell size for the 9 CHO sub-clones they studied.…”

“…To obtain an increased volumetric productivity without increasing cell density, an increase in specific productivity (qp) is needed. For this, several strategies have been described, including hyperosmotic pressure 92 , cell cycle arrest 53 , and mild hypothermia 57 . In these studies, the increase in qp was found to be correlated with an increase in cell size.…”