Gene-expression profiles for five key glycosylation genes for galactose-fed CHO cells expressing recombinant IL-4/13 cytokine trap.
Recombinant protein glycosylation profiles have been shown to affect the in-vivo half-life, and therefore the efficacy and economics, for many therapeutics. While much research has been conducted correlating the effects of various stimuli on recombinant protein glycosylation characteristics, relatively little work has examined glycosylation-related gene-expression profiles. In this study, the effects of galactose feeding on the gene-expression profiles for five key glycosylation-related genes were determined for Chinese hamster ovary cells producing a recombinant IL-4/13 cytokine trap fusion. The genes investigated were sialidase, a putative alpha2,3-sialyltransferase, CMP-sialic acid transporter, beta1,4-galactosyltransferase, and UDP-galactosyltransferase. Additionally, the sialic acid content (sialylation) of the recombinant protein was examined. The peak sialic acid content of the IL-4/13 cytokine trap fusion protein was observed to be similar for the control and galactose-fed cultures. The gene-expression profiles for four of the glycosylation genes were observed to be sensitive to the glucose concentration and not significantly different for the control and galactose-fed cultures prior to glucose depletion. However, the sialidase gene-expression profiles were different for the control and galactose-fed cultures. The sialidase gene-expression profile increased significantly for the galactose-fed cultures prior to glucose depletion, whereas for the control cultures, the sialidase gene-expression profiles did not increase until the late stationary phase. The intracellular sialidase enzyme activity decreased exponentially with time for the control cultures; however, for the galactose-fed cultures, the intracellular sialidase enzyme activity decreased initially and then remained relatively high compared to the control cultures. These results indicate that the galactose feeding may increase the potential for desialylation, which offsets any improvements in the sialylation rate due to increased substrate levels. Thus, galactose feeding is an unnecessary expense for the production of the IL-4/13 cytokine trap fusion protein in a batch process.
Clark
Kevin J-R
KJ
Department of Chemical Engineering, 112 Biosystems Research Complex, Clemson University, Clemson, South Carolina 29634-0905, USA.
Griffiths
Jennifer
J
Bailey
Kevin M
KM
Harcum
Sarah W
SW
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005690:Galactose / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology*; D006031:Glycosylation; D018793:Interleukin-13 / Q000235:genetics / Q000378:metabolism*; D015847:Interleukin-4 / Q000235:genetics / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014166:Transferases / Q000378:metabolism*
D018793:Interleukin-13; D011993:Recombinant Fusion Proteins; D015847:Interleukin-4; D014166:Transferases; D019158:N-Acetylneuraminic Acid; D005690:Galactose