Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK‐21 cells

Abstract

The native structures of the Asn‐linked oligosaccharides and the O‐glycans at Ser126 of human erythropoietin expressed from recombinant BHK cells have been elucidated. Enzymatically released N‐glycans were studied by methylation analyses, fast‐atom‐bombardment mass spectrometry as well as one‐ and two‐dimensional 1H‐NMR spectrometry at 600 MHz. Many (82.7%) were found to be tetraantennary N‐acetyllactosamine‐type (22.8% with one, 3.6% with two and 0.4% with three N‐acetyllactosamine repeats) being tetrasialylated (41%), trisialylated (29.6%) and disialylated (12.2%). A few (9.7%; 4.1% 2,4‐branched, 5.6% 2,6‐branched) of the chains were triantennary (5.4% trisialyl, 4.3% disialyl) and 4.6% were of the disialyl diantennary type. Almost all of the innermost GlcNAc residues were α1–6 fucosylated and NeuAc was exclusively α2–3 linked to Galβ1–4GlcNAc‐R; 60% of the protein was found to be O‐glycosylated at Ser126; structures were monosialylated (70%) or disialylated (30%) forms of the Galβ1–3GalNAc core type. Glycosylation patterns at individual Asn‐Xaa‐Thr/Ser sites were determined by analytical high‐pH anion‐exchange chromatography with pulsed amperometric detection. Only tetraantennary chains with 0–3 N‐acetyllactosamine repeats were detected at Asn38 and Asn83, while almost all of the di‐ and triantennary oligosaccharides were attached to Asn24. Batch analysis of different preparations of recombinant erythropoietin revealed the high reproducibility of the production procedure. Structures containing terminal GalNAc–GlcNAc were detected in small amounts in a few batches.