N‐ and O‐linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte‐macrophage colony‐stimulating factor secreted by a Chinese hamster ovary cell line

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Abstract
GM‐CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM‐CSF from an engineered Chinese hamster ovary cell line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N‐glycosylation sites occupied. Minor low molecular mass forms completely lacked N‐linked oligosaccharides but contained 1–3 O‐linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex‐HexNAc units (Δm 365 Da), were detected in MALDI‐TOF MS spectra of the desialylated GM‐CSFs. MALDI‐TOF MS and HPAEC‐PAD analysis indicated the presence of predominantly tri‐ and tetraantennary N‐linked oligosaccharide chains with and without N‐acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal α1–6‐linked fucose. The oligosaccharide patterns of both GM‐CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N‐glycans were found α2–3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM‐CSF form more than 90% of the Asn37 were occupied by N‐glycans. O‐glycosylation at the N‐terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O‐glycosylated glycoprotein form. In the triply modified GM‐CSF molecules, Ser5 was additionally O‐glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N‐glycosylation site occupied in the preparation derived from suspension culture. ESI‐MS and MALDI‐MS analysis of endoproteolytically cleaved peptides as well as MALDI‐TOF MS of the intact glycoprotein demonstrated the N‐ and C‐termini integrity of the GM‐CSF preparations.

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