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|Title:||Profiling of N-glycosylation gene expression in CHO cell fed-batch cultures|
|Keywords:||Chinese hamster ovary (CHO)|
|Citation:||Wong, D.C.F., Wong, N.S.C., Goh, J.S.Y., May, L.M., Yap, M.G.S. (2010-10-15). Profiling of N-glycosylation gene expression in CHO cell fed-batch cultures. Biotechnology and Bioengineering 107 (3) : 516-528. ScholarBank@NUS Repository. https://doi.org/10.1002/bit.22828|
|Abstract:||One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N-glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N-glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed-batch culture of a CHO cell line producing recombinant human interferon-γ (IFN-γ). Of the 24 N-glycosylation genes examined, 21 showed significant up-or down-regulation of gene expression as the fed-batch culture progressed from exponential, stationary and death phase. As the fed-batch culture progressed, there was also an increase in less sialylated IFN-γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN-γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed-batch strategy appears to need a 0.5mM glutamine threshold to maintain similar N-glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible "bottlenecks" or "compromised" pathways in N-glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. © 2010 Wiley Periodicals, Inc.|
|Source Title:||Biotechnology and Bioengineering|
|Appears in Collections:||Staff Publications|
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