Effect of Lipids on Recombinant Interferon-γ Glycosylation

Batch cultures of Chinese hamster ovary cells expressing human interferon-gamma (IFN-gamma) were supplemented with the lipids dolichol or dolichyl phosphate. End of batch samples were analysed by micellar electrokinetic capillary electrophoresis to examine changes in glycosylation site occupancy. A detailed analysis of carbohydrate structures at each site was performed using matrix-assisted laser desorption/ionisation mass spectrometry.

Cultures that had received a single supplement of dolichol or dolichyl phosphate at the start of culture exhibited a 16 % increase in the proportion of fully glycosylated (2N) IFN-gamma, with a concomitant decrease in the proportion of non-glycosylated IFN-gamma. The proportions of molecules with a single site occupied (1N) remained relatively constant. However, the effects of the lipids on glycosylation appear to be influenced by other conditions, such as cell growth. When cultures showing sub-optimal growth received repeated supplements of dolichol during growth phase, a similar 16 % increase in the 2N site occupancy variant was observed. Whereas repealed dolichyl phosphate supplements produced a 47 % increase in fully glycosylated IFN-gamma. Again the proportions of 1N remained relatively constant. Cultures with normal growth characteristics showed no changes in the proportions of site occupancy variants after receiving repeated lipid supplements. In most cases, lipid supplements had no effect on the types of N-glycan structure present at each glycosylation site. A possible exception to this was after repeated dolichol supplements, when the IFN-gamma produced exhibited an increase in complex structures at the Asn(97) glycosylation site.