Caspase activation, sialidase release and changes in sialylation pattern of recombinant human erythropoietin produced by CHO cells in batch and fed-batch cultures

Abstract

The activation of caspases represents a crucial turning point during a batch or a fed-batch culture of mammalian cells. It not only affects the quantity but also the quality of the recombinant glycoprotein produced. In this study, the activation of various caspases, the release of intracellular sialidase and the changes in sialylation pattern of a recombinant product, erythropoietin (EPO), in the culture medium were analyzed in both batch and fed-batch cultures. In both setups, all caspase activities peaked at the culture time point at which decline of cell viability was most pronounced. In addition, the release of intracellular lactate dehydrogenase (LDH) was also tracked during these cultures. The increase in LDH activity in the medium coincided with the increase of intracellular caspase activities, the release of sialidase and the observed decline in cell viability, suggesting that the LDH activity in the medium can be used as an indirect indicator of apoptotic cell death in bioreactors. Isoelectric focusing (IEF) coupled with double blotting was employed to analyze the changes in sialylation pattern of the recombinant EPO. This assay resulted in a prompt resolution of secreted EPO isoforms in a time course format. IEF profile of batch culture showed relatively consistent product sialylation compared to fed-batch culture, which showed gradual band shifts towards the isoforms with fewer sialic acid as the culture progressed. These data provided a guideline for the optimal time point to terminate the culture and collect products in batch and fed-batch cultures.

Fig. 1

Cell viability and caspase activation during batch and fed-batch cultures. (A) Viable cell density, cell viability and intracellular caspase-2, −3, −5, −6, −8, −9 like activity profiles of batch culture. (•) Viable cell density; (○) cell viability; ( ) caspase-2 like activity; (■) caspase-3 like activity; ( ) caspase-5 like activity; (▴) caspase-6 like activity; ( × ) caspase-8 like activity; (▵) caspase-9 like activity. (B) Viable cell density, cell viability and intracellular caspase-2, −3, −5, −6, −8, −9 like activity profiles of fed-batch culture. (C) Western blot analysis of caspase-3 activation during the course of a batch culture. (D) Western blot analysis of caspase-3 activation during the course of a fed-batch culture

Fig. 2

Release of cellular sialidase and LDH in batch and fed-batch cultures. (A) Cell viability, extracellular sialidase activity and LDH activity profiles of the same batch culture shown in Fig. 1A. (○) Cell viability; (▴) LDH activity; (■) sialidase activity. (B) Cell viability, extracellular sialidase activity and LDH activity profiles of the same fed-batch culture shown in Fig. 1B

Fig. 3

Productivity of EPO in batch and fed-batch cultures. (A) Volumetric EPO yield, intracellular caspase-3 like activity and cell viability profiles of the same batch culture shown in Fig. 1A. (•) EPO concentration; (■) caspase-3 like activity; (○) cell viability. (B) Volumetric EPO yield, intracellular caspase-3 like activity and cell viability profiles of the same fed-batch culture shown in Fig. 1B. (•) EPO concentration; (■) caspase-3 like activity; (○) cell viability

Fig. 4

Changes in sialylation pattern of EPO in batch and fed-batch cultures analyzed with IEF. (A) IEF profile of secreted EPO in the batch culture. (B) IEF profile of secreted EPO in the fed-batch culture. (C) Band intensity spectrum of a batch culture sample obtained using “AIDA 1D-Evaluation” software from Fuji

Fig. 5

Sialylation quality profile of EPO in batch and fed-batch cultures and its association with cell viability. (▵) Sialylation quality score of the batch culture; ( ) sialylation quality score of the fed-batch culture; (▴) cell viability of the batch culture; (■) cell viability of the fed-batch culture. The numbers shown here is not an absolute quantification of the sialic acid content of EPO but a relative comparison of sialylation between EPO products at various time points of both batch and fed-batch cultures