N-GLYCANS OF RECOMBINANT HUMAN INTERFERON-GAMMA CHANGE DURING BATCH CULTURE OF CHINESE-HAMSTER OVARY CELLS

A recombinant Chinese hamster ovary (CHO) cell line making human inter feron-gamma (IFN-gamma) was grown in 12-L stirred tank fermenters in t hree batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition to cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate struct ures attached to each glycosylation site of IFN-gamma was achieved usi ng matrix-assisted laser desorption mass spectrometry (MALDI-MS) in co mbination with exoglycosidase array sequencing. Complex biantennary ol igosaccharides (particularly Gal(2)GlcNAc(4)Man(3) which was core alph a 1-6 fucosylated at Asn(25) but not at Asn(97)) were most prevalent a t both glycosylation sites. However, considerable microheterogeneity a rising from the presence of triantennary and truncated glycan structur es was also observed. The proportion of the dominant core glycan struc ture (Gal(2)GlcNAc(4)Man(3) +/- Fuc(1)) decreased by 15-26% during bat ch culture, with increases in the proportion of oligomannose and trunc ated glycans over the same time period. Prolonged culture resulting fr om an extended lag phase led to further accumulation of oligomannose a nd truncated structures, reaching up to 52% of total glycans attached to Asn(97) by 240 h of culture. The implications of these glycosylatio n changes for optimizing the time for harvesting cell cultures, and fo r the clearance of recombinant therapeutic products in vivo are discus sed. (C) 1995 John Wiley & Sons, Inc.