Profiling of N-glycosylation gene expression in CHO cell fed-batch cultures.
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-gamma (IFN-gamma). 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-gamma glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN-gamma. 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 0.5 mM 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.
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01, Singapore 138668, Singapore.
Danny Chee Furng
DC
Wong
Niki Soo Ching
NS
Wong
John Soo Yang
JS
Goh
Lee May
LM
May
Miranda Gek Sim
MG
Yap
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D020869:Gene Expression Profiling; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000096:biosynthesis*; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D006023:Glycoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007371:Interferon-gamma; D016695:Glycosyltransferases; D019158:N-Acetylneuraminic Acid