ISSN:
0006-3592
Keywords:
micellar electrokinetic capillary chromatography
;
capillary isoelectric focusing
;
Chinese hamster ovary
;
interferon-gamma
;
perfusion culture
;
glycosylation
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Chinese hamster ovary cells producing recombinant human interferon-γ were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized-bed bioreactor, reaching a maximum cell density in excess of 3 × 107 cells (mL microcarrier)-1 at a specific growth rate (μ) of 0.010 h-1. During establishment of the culture, the N-glycosylation of secreted recombinant IFN-γ was monitored by capillary electrophoresis of intact IFN-γ proteins and by HPLC analysis of released N-glycans. Rapid analysis of IFN-γ by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN-γ (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-γ 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-γ 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-γ during culture, with a concomitant decrease in the proportion of monosialylated and neutral N-glycans. Comparative analyses of IFN-γ 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. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 596-607, 1998.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
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