Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells.
Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, South Korea.
Yun Hee
YH
Sung
Yeon Jung
YJ
Song
Seung Wook
SW
Lim
Joo Young
JY
Chung
Gyun Min
GM
Lee
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002087:Butyrates / Q000494:pharmacology*; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012964:Sodium / Q000494:pharmacology*; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000235:genetics*
D002087:Butyrates; D011994:Recombinant Proteins; D013926:Thrombopoietin; D012964:Sodium