Erythropoietin Produced in a Human Cell Line (Dynepo) Has Significant Differences in Glycosylation Compared with Erythropoietins Produced in CHO Cell Lines

Abstract

Recombinant human erythropoietin has been used to treat anemia associated with chronic renal disease. This paper provides a comprehensive comparative analysis of Dynepo and three other commercial erythropoiesis stimulating agents, Eprex, NeoRecormon and Aranesp. We found significant differences in the type, levels and amount of O-acetylation of sialic acids. Sialic acids and O-acetylation present provide protection from clearance from circulation. Aranesp had up to six O-acetyl groups attached to the sialic acids. Eprex and NeoRecormon had only minor amounts of O-acetylation while Dynepo had none. Dynepo had no Neu5Gc, which is potentially immunogenic for humans. Dynepo contained the least amount of disialylated and Aranesp the highest amount of tetrasialylated glycans. NeoRecormon and Eprex contained more trisialylated, but less tetrasialylated glycans than Dynepo and Aranesp. Dynepo had the highest amount of tetraantennary glycans and the lowest amounts of triantennary glycans with a β1-6-GlcNAc linkage. All the samples contained poly-N-acetyl-lactosamine repeats with Dynepo having the least. The major N-acetyl-lactosamine extensions in Dynepo and Aranesp were on biantennary glycans, whereas in NeoRecomon and Eprex they were on triantennary glycans. The sLe(x) epitope was only detected in Dynepo.

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).…”

“…4,8,10,43,44 These modifications were mainly observed in highly sialylated glycans and include acetylation of sialic acids, N-acetylneuraminic (Neu5Ac) / N-glycolylneuraminic acid (Neu5Gc) variation, and elongation of the glycan chains because of the presence of LacNAc repeats. Up to three modifications occurred per glycan.…”

“…This phenomenon has not been extensively reported and there are only a few studies in the literature dealing with the presence of Neu5Gc in rHuEPO. 8,10,44,53,60 Even though Neu5Gc is a common sialic acid in most mammals, it has been demonstrated to be absent in healthy humans and only small amounts have been found in some tumors and meconium. 61 The CE-MS method succeeded in detecting this heterogeneity of acidic monosaccharides without removing the sialic acid from the glycan chain by acid hydrolysis as it has been commonly reported.…”

“…61 The CE-MS method succeeded in detecting this heterogeneity of acidic monosaccharides without removing the sialic acid from the glycan chain by acid hydrolysis as it has been commonly reported. 8,53,62 As reviewed lately, the LC-MS detection of sialic acids and its heterogeneous subtypes is challenging. 63 Mass spectrometric results indicate that the variation occurred at the glycan level as shown in Figure 3B and Supporting Information (Figures S1.B and S1.D).…”

Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization–mass spectrometry

“…Detailed monitoring and controlling glycosylation is essential in biopharmaceutical development and quality control of drugs that are glycosylated (e.g., erythropoietin) [3,4].…”