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

Abstract

The native structures of the Asn-linked oligosaccharides and the 0-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 'H-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 Nacetyllactosamine 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 a1 -6 fucosylated and NeuAc was exclusively a2-3 linked to Galljl-4GlcNAc-R; 60% of the protein was found to be 0-glycosylated at Ser126; structures were monosialylated (70%) or disialylated (30%) forms of the Galljl -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.Human erythropoietin (EPO) is the principle regulator of hematopoiesis and stimulates the differentiation of the erythroid progenitor cells into mature erythrocytes (Jacobson et al., 1975). The hormone is produced mainly in the adult kidneys and fetal liver. EPO exerts its effect by binding to receptor proteins on the surface of erythroid progenitor cells . Clinical trials with EPO have revealed its great potential for the treatment of chronic anemia caused by impaired production and loss of the hormone due to and during treatment of renal failure (Winearles et al., 1986).

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

“…58,59 For that reason, rHuEPO expressed in CHO cells has been reported to contain between 1 and 1.5% of Neu5Gc residues relative to the total sialic acid content, while EPO products engineered by gene-activation in human cells contain no Neu5Gc. 8,57,60 Our CE-MS method detected the presence of Neu5Gc, instead of Neu5Ac, in some N-glycans from rHuEPO. In some cases, this sialic acid variation occurred simultaneously with the acetylation of glycans previously discussed ( Figure 3B).…”

Sequencing of tri- and tetraantennary N-glycans containing sialic acid by negative mode ESI QTOF tandem MS

“…For the triantennary N-glycan with the 6-linked antenna branched, the internal fragment ion D formed by loss of the 3-antenna, together with the chitobiose core (B 5 /Y 3␣ ), was identified as the important diagnostic ion for the branching and antenna composition. For detection of core fucose, the cross-ring fragment ions 0,2 A and 2,4 A, resulting from the reducing termini of N-glycans, show the mass difference between fragment ions 0,2 A and 2,4 A of 206 Da with the core-Fuc and 60 Da without it.…”

“…After detachment of the total N-glycans with polypeptide N-glycanase, the oligosaccharide mixture was subjected to ion exchange chromatography on Mono Q [2] and the individual charged oligosaccharide fractions were pooled separately (asialo-, monosialo to pentasialo fraction). The trisialylated fraction was further resolved by HPLC on NH 2 -bonded phase.…”

“…Besides, the expression of a certain oligosaccharide epitope may be restricted to just one of the antennae present. In order to carry on a complete characterization of complex type N-glycan mixtures obtained from glycoproteins after release by Nglycanase, combined chromatographic, mass spectrometric and nuclear magnetic resonance spectroscopic approach can be mandatory, as demonstrated in the case of human erythropoietin (hEPO), a glycoprotein containing bi-, tri-and tetraantennary N-glycan structures [2]. Its biological activity in terms of the clearance rate was positively correlated with the ratio of tetra-to biantennary N-glycans, where the hEPO with wellbranched tetraantennary glycans remained at higher levels in the plasma [3].…”

Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines

“…EPO is a well-characterized pharmaceutical protein product of cultured mammalian cells (Takeuchi et al 1989;Yamaguchi et al 1991;Nimtz et al 1993;Schlenke et al 1999). To examine if the proliferation-controlled cells could be used for recombinant gene expression, LT1 cells were genetically modified using an adenoviral vector carrying a recombinant EPO expression cassette.…”