Monitoring recombinant human interferon-gamma N-glycosylation during perfused fluidized-bed and stirred-tank batch culture of CHO cells

中国仓鼠卵巢细胞 糖基化 重组DNA 聚糖 微载波 干扰素γ 化学 色谱法 细胞培养 N-连接糖基化 生物化学 分子生物学 生物 体外 糖蛋白 细胞 基因 受体 遗传学
作者
Merlin H. Goldman,David C. James,Mark Harcus Rendall,A. P. Ison,M. Hoare,Alan T. Bull
出处
期刊:Biotechnology and Bioengineering [Wiley]
卷期号:60 (5): 596-607 被引量:76
标识
DOI:10.1002/(sici)1097-0290(19981205)60:5<596::aid-bit10>3.0.co;2-5
摘要

Chinese hamster ovary cells producing recombinant human interferon-gamma were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized-bed bioreactor, reaching a maximum cell density in excess of 3 x 10(7) cells (mL microcarrier)-1 at a specific growth rate (mu) of 0.010 h-1. During establishment of the culture, the N-glycosylation of secreted recombinant IFN-gamma was monitored by capillary electrophoresis of intact IFN-gamma proteins and by HPLC analysis of released N-glycans. Rapid analysis of IFN-gamma by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN-gamma (two Asn sites occupied, one Asn site occupied and nonglycosylated) in under 10 min per sample; the relative proportions of these variants remained constant during culture. Analysis of IFN-gamma by capillary isoelectric focusing resolved at least 11 differently sialylated glycoforms over a pI range of 3.4 to 6.4, enabling rapid quantitation of this important source of microheterogeneity. During perfusion culture the relative proportion of acidic IFN-gamma proteins increased after 210 h of culture, indicative of an increase in N-glycan sialylation. This was confirmed by cation-exchange HPLC analysis of released, fluorophore-labeled N-glycans, which showed an increase in the proportion of tri- and tetrasialylated N-glycans associated with IFN-gamma during culture, with a concomitant decrease in the proportion of monosialylated and neutral N-glycans. Comparative analyses of IFN-gamma produced by CHO cells in stirred-tank culture showed that N-glycan sialylation was stable until late in culture, when a decline in sialylation coincided with the onset of cell death and lysis. This study demonstrates that different modes of capillary electrophoresis can be employed to rapidly and quantitatively monitor the main sources of glycoprotein variation, and that the culture system and operation may influence the glycosylation of a recombinant glycoprotein.
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