Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells

Abstract

Recombinant human erythropoietin (rHuEPO) is used abundantly in the clinic to stimulate red blood cell growth in anaemic patients. The efficacy of the drug depends strongly on the extent of sialylation of its carbohydrate moiety. Prompted by conflicting literature reports on the issue, we reinvestigated the structures of the intact sialylated carbohydrate chains of rHuEPO expressed in Chinese hamster ovary (CHO) cells. The asparagine-linked oligosaccharides were released from rHuEPO with N-glycanase and fractionated by anion-exchange chromatography. The O-linked oligosaccharides were released under alkaline borohydride conditions. The primary structures of the major sialylated N- and O-type oligosaccharides were identified by 500-MHz 1H-NMR spectroscopy, supported by data from composition analysis, methylation analysis, low- and high-pH anion-exchange chromatography, and fast atom bombardment-mass spectrometry. The most abundant N-linked oligosaccharides in CHO cell-derived rHuEPO were found to be di-antennary, 2,4-branched tri-antennary, 2,6-branched tri-antennary and tetra-antennary chains (in the ratio of 7:6:5:82), with the latter containing between zero and three repeating N-acetyllactosamine units, in well-defined branches. The major (> 95%) di-, tri- and tetra-antennary structures are fully sialylated, i.e. they have two, three and four sialic acid residues, respectively, linked exclusively alpha(2-->3) to galactose residues. The majority (> 95%) of N-linked structures contain alpha(1-->6)-linked fucose at the proximal GlcNAc residue. The O-type mono- and disialyl oligosaccharides were characterized as a linear tri- and a branched tetra-saccharide, respectively.

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

“…Furthermore, bisecting N-acetylglucosamine residues have never been detected in structural studies of the N-glycans of rEPOs and other glycoproteins synthesized in CHO cells. Figure 6 shows the N-glycan structures found in SpB, as well as other structures, which are known from previous studies (Rice et al, 1992;Watson et al, 1994;Hokke et al, 1995;Kanazawa et al, 1999) to be commonly present in rEPOs synthesized in CHO cells.…”

“…The structures of the oligosaccharides in individual Amide-80 HPLC fractions were then analysed by MALDI mass spectrometry to give the structures proposed in Table V. These structures were deduced from their molecular masses, their elution profiles on DEAE and Amide-80 HPLC, and from published data (Rice et al, 1992;Watson et al, 1994;Hokke et al, 1995;Kanazawa et al, 1999).…”

“…Like other glycoprotein hormones, EPO exists as mixtures of a multitude of isoforms that differ mainly in their glycosylation (Storring, 1992). Thus, each of the three N-glycosylation sites may be occupied by a range of different glycans, with some heterogeneity also among the glycans at the single O-glycosylation site (Sasaki et al, 1987;Takeuchi et al, 1988;Tsuda et al, 1988;Nimtz et al, 1993;Watson et al, 1994;Hokke et al, 1995). The isoform composition of a particular preparation of EPO depends on its source.…”

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

“…56,57 Contrary to human cells, CHO cells do not express sialyl-α-2,6-transferase, α-1,3/4-fucosyltransferase and bisecting N-acetylglucosamine transferase. 47 However, these cells contain the enzyme responsible for N-glycolylneuraminic acid synthesis (CMP-N-acetylneuraminic acid hydroxylase [CMAH]) that is not present in humans due to an internal frame shift mutation in the CMAH human gene. 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.…”

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

“…Host-cell type and culture conditions have been shown to be the most critical ones (Goto et al 1988;Goochee and Monica 1990;Jenkins et al 1996;Zhang et al 2002;Tomiya et al 2003;Yoon et al 2005). In particular, various elements of the extracellular environment, including the configuration of the production system (Watson et al 1994;Cabrera et al 2005;Lipscomb et al 2005;Spearman et al 2005) and the composition of the culture medium like ammonia concentration (Andersen and Goochee 1995;Borys et al 1994;Gawlitzek et al 1998;Jenkins and Curling 1994;Grammatikos et al 1998;Yang and Butler 2000a, b;Chen and Harcum 2005), glucosamine supplementation (Yang and Butler 2002), lipid availability , Na-butyrate addition (Lamotte et al 1999;Sung et al 2005) are able to affect protein glycosylation by potentially inducing changes in cell metabolism or favouring enzymatic product degradation.…”