N‐glycosylation microheterogeneity and site occupancy of an Asn‐X‐Cys sequon in plasma‐derived and recombinant protein C

Human protein C (hPC) is glycosylated at three Asn‐X‐Ser/Thr and one atypical Asn‐X‐Cys sequons. We have characterized the micro‐ and macro‐heterogeneity of plasma‐derived hPC and compared the glycosylation features with recombinant protein C (tg‐PC) produced in a transgenic pig bioreactor from two animals having approximately tenfold different expression levels. The N‐glycans of hPC are complex di‐ and tri‐sialylated structures, and we measured 78% site occupancy at Asn‐329 (the Asn‐X‐Cys sequon). The N‐glycans of tg‐PC are complex sialylated structures, but less branched and partially sialylated. The porcine mammary epithelial cells glycosylate the Asn‐X‐Cys sequon with a similar efficiency as human hepatocytes even at these high expression levels, and site occupancy at this sequon was not affected by expression level. A distinct bias for particular structures was present at each of the four glycosylation sites for both hPC and tg‐PC. Interestingly, glycans with GalNAc in the antennae were predominant at the Asn‐329 site. The N‐glycan structures found for tg‐PC are very similar to those reported for a recombinant Factor IX produced in transgenic pig milk, and similar to the endogenous milk protein lactoferrin, which may indicate that N‐glycan processing in the porcine mammary epithelial cells is more uniform than in other tissues.