Caspase activation, sialidase release and changes in sialylation pattern of recombinant human erythropoietin produced by CHO cells in batch and fed-batch cultures.
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.
Chuan
Kok Hwee
KH
Bioprocessing Technology Institute, Agency for Science, Technology and Research, 20 Biopolis Way, 06-01 Centros, Singapore, 138668, Singapore.
Lim
Sing Fee
SF
Martin
Laurent
L
Yun
Chee Yong
CY
Loh
Sophia O H
SO
Lasne
Francoise
F
Song
Zhiwei
Z