Knowing the glycosylation machinery of mammary gland cells: Potential and drawbacks for the expression of proteins of pharmaceutical interest

N-glycosylation analysis of recombinant human Erythropoietin (rhEPO) obtained by adenoviral transduction of goat mammary gland epithelial cells (GMGE) rhEPO-GMGE and in the milk of goats (rhEPO-Lc) was carried out by a combination of normal-phase HPLC (Amide-80) and ion exchange chromatography of the 4ABA labeled, enzymatically released N-glycans and further characterized by MALDI, ESI-MS and LC/MS. The most abundant N-glycans of rhEPO-GMGE are the monosialylated multiantennary core-fucosylated type, but fucosylation was also found in outer arms. However, rhEPO-Lc showed low branched, core-fucosylated, N-glycans. Here the charged N-glycans were found to be mostly a2,6-monosialylated with Neu5Ac or Neu5Gc at a ratio of 1:1, in contrast with the Nglycans from rhEPO produced in GMGE cells, where the charged glycans display the Neu5Ac. An important finding was the presence in rhEPO-Lc of biantennary N-glycans with lactosediamine (GalNAc-GlcNAc) ending arms that can be either neutral or sialylated, which is poorly represented in rhEPO-GMGE. This type of non-reducing terminal has not been found in rhEPO-CHO. These features differentiate the recombinant EPO expressed in the goat mammary gland from the classical EPO expressed in CHO cells, where the N-glycans are mostly fully sialylated multiantennary structures. These results emphasize that the difference between N-glycan populations of a given glycoprotein are sensitive to the cell-type and cell environment where they are cultivated.