2D-LC Analysis of BRP 3 Erythropoietin N-Glycosylation using Anion Exchange Fractionation and Hydrophilic Interaction UPLC Reveals Long Poly-N-Acetyl Lactosamine Extensions

Abstract

Post translational modifications, in particular glycosylation, represent critical structural attributes that govern both the pharmacodynamic and pharmacokinetic properties of therapeutic glycoproteins. To guarantee safety and efficacy of recombinant therapeutics, characterization of glycosylation present is a regulatory requirement. In the current paper, we applied a multidimensional strategy comprising a shallow anion exchange gradient in the first dimension, followed by analysis using the recently introduced 1.7 μm HILIC phase in the second dimension for the comprehensive separation of complex N-glycans present on the European Biological Reference Preparation (BRP) 3 erythropoietin standard. Tetra-antennary glycans with multiple sialic acids and poly-N-acetyl lactosamine extensions were the most abundant oligosaccharides present on the molecule. Site-specific glycan analysis was performed to examine microheterogeneity. Tetra-antennary glycans with up to four sialic acids and up to five poly-N-acetyl lactosamine extensions were observed at asparagine 24 and 83, while bi-antennary glycans were the major structures at asparagine 38. The combined AEC × UPLC HILIC allows for the rapid and comprehensive analysis of complex N-glycosylation present on therapeutic glycoproteins such as BRP3 erythropoietin.

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

“…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.…”

“…In that study, successful detection of tetra-antennary glycans with extensive LacNAc repeats was based on a combination of weak anion exchange chromatography (AEC) and HILIC that allowed for the first time the detection of up to five LacNAc repeats. 4 LC-MS/MS Analysis of N-Glycans from rHuEPO. Underivatized, nonreduced N-glycans of rHuEPO were also analyzed using online porous graphitized carbon (PGC) LC-MS/MS to compare its ability to glycoprofile complicated glycan mixtures with that of CE-MS. Table S.III in the Supporting Information shows a list of the glycans detected with PGC LC-MS/MS.…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…[115][116][117] HILIC tolerates neutral and acidic glycans, regardless of whether they are labeled with fluorescent tags. 118 Typical equilibration conditions in the HILIC HPLC of mono-and oligosaccharides and glycopeptides are 10 -25% water in acetonitrile with a low concentration of acid or salt (mostly below 100 mM).…”

“…[137][138][139] Recently 2-AB labeled oligosaccharides have also been separated by multi-separation methods using a combination of ion-exchange and HILIC. 115 A combination of online 2D LC systems was found to be useful in the separation of glycopeptides in protease digest mixtures. 140 …”

Capillary electrophoresis in the N-glycosylation analysis of biopharmaceuticals

“…subject to 307 derivatization, purification and exoglycosidase-array-308 based glycan sequencing). This method was successfully 309 applied to the analysis of heavily-sialylated biopharma-310 ceuticals (e.g., erythropoietin) [42]. Extra charges on 311 glycans can be caused by other groups (e.g., phosphor-312 ylation), in which case immobilized metal affinity chro-313 matography (IMAC) was successfully utilized for their 314 partitioning before the application of the exoglycosidase-315 based sequencing [43].…”