Physiological responses of CHO cells to repetitive hydrodynamic stress

Abstract

A majority of the previous investigations on the hydrodynamic sensitivity of mammalian cells have focused on lethal effects as determined by cell death or lysis. In this study, we investigated the effect of hydrodynamic stress on CHO cells in a fed-batch process using a previously reported system which subjects cells to repetitive, high levels of hydrodynamic stress, quantified by energy dissipation rate (EDR). The results indicated that cell growth and monoclonal antibody production of the test cells were very resistant to the hydrodynamic stress. Compared to the control, no significant variation was observed at the highest EDR tested, 6.4 x 10(6) W/m(3). Most product quality attributes were not affected by intense hydrodynamic stress either. The only significant impact was on glycosylation. A shift of glycosylation pattern was observed at EDR levels at or higher than 6.0 x 10(4) W/m(3), which is two orders of magnitude lower than the EDR where physical cell damage, as measured by lactate dehydrogenase release, was observed. While not as extensively investigated, a second monoclonal antibody produced in a different CHO clone exhibited the same glycosylation change at an intensive EDR, 2.9 x 10(5) W/m(3). Conversely, a low EDR of 0.9 x 10(2) W/m(3) had no effect on the glycosylation pattern. As 6.0 x 10(4) W/m(3), the lowest EDR that triggers the glycosylation shift, is about one order of magnitude higher than the estimated, maximum EDR in typically operated, large-scale stirred tank bioreactors, further studies in a lower EDR range of 1 x 10(3)-6.0 x 10(4) W/m(3) are needed to assess the glycosylation shift effect under typical large-scale bioreactor operation conditions. Follow-up studies in stirred tanks are also needed to confirm the glycosylation shift effect and to validate the repetitive hydrodynamic stress model.

Scale-down studies for assessing the impact of different stress parameters on growth and product quality during animal cell culture

“…The loop was essentially as described earlier (Godoy-Silva et al, 2009a;2009b) antibody product quality was assessed using a Genentech protocol in a similar set of ways to that set out above. It should be noted that with the techniques employed to assess quality, it would be difficult to detect any changes in charge profiles associated with sialylation.…”

“…Consistency of glycoform is a major consideration when assessing protein quality for therapeutic purposes (Jenkins, 2007;Godoy-Silva et al, 2009a;2009b). As can be seen, the small variation in glycoforms between the differing experimental conditions to which the cells were exposed is quite random and typical of that found in industrial historical data.…”

“…Overall, it was decided to try circulation with a syringe pump as was used in the previous work by Godoy-Silva et al, (2009a;2009b) when using the extensional flow device.…”

“…To further explore such issues, Chalmers and co-workers (Godoy-Silva et al, 2009a, 2009b ). However, in the first case, cell growth was inferior compared to controls without recirculation (Godoy-Silva et al, 2009a); and in the second, it was product quality (Godoy-Silva et al, 2009b).…”

“…The aim was to clarify the earlier work of Godoy-Silva et al (2009a;2009b) on the impact of laminar stresses on process performance and product quality using two Genentech production CHO cells lines. In addition, further studies with these two lines were conducted in a bioreactor agitated by dual Rushton turbines at turbulent mean specific energy rates, T ε up to 1000 W/m 3 .…”