Metabolic shifts do not influence the glycosylation patterns of a recombinant fusion protein expressed in BHK cells

BHK-21 cells expressing a human IgG-IL2 fusion protein, with potential appl ication in tumor-targeted therapy, were grown under different nutrient cond itions in a continuous system for a time period of 80 days. At very low-glu cose (< 0.5 mM) or glutamine (< 0.2 mM) concentrations, a shift toward an e nergetically more efficient metabolism was observed. Cell-specific producti vity was maintained under metabolically shifted growth conditions and at th e same time an almost identical intracellular ATP content, obtained by in v ivo P-31 NMR experiments, was observed. No significant differences in the o ligosaccharide structures were detected from the IgG-IL2 fusion protein pre parations obtained by growing cells under the different metabolic states. B y using oligosaccharide mapping and MALDI/TOF-MS, only neutral diantennary oligosaccharides with or without core alpha 1-6-linked fucose were detected that carried no, one or two beta 1-4-linked galactose. Although the O-link ed oligosaccharide structures that are present in the IL2 moiety of the pro tein were studied with less detail, the data obtained from the hydrazinolys is procedure point to the presence of the classical NeuAc alpha 2-3Gal beta 1-3GalNAc structure. Here, it is shown that under different defined cellul ar metabolic states, the quality of a recombinant product in terms of O- an d N-linked oligosaccharides is stable, even after a prolonged cultivation p eriod. Moreover, unaffected intracellular ATP levels under the different me tabolic states were observed. (C) 2000 John Wiley & Sons, Inc.