Understanding and characterizing protein therapeutic glycosylation is important with growing evidence that glycosylation impacts biological efficacy, pharmacokinetics, and cellular toxicity. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an effective tool for separating the glycans present on glycoprotein therapeutics. A new operating mode for HPAE-PAD systems, Dual Eluent Generation Cartridge (Dual EGC) mode, is available to support the analysis of complex carbohydrates. This mode of operation replaces the manual preparation of the sodium hydroxide/sodium acetate eluents required for analyzing complex carbohydrates by HPAE-PAD with electrolytic generation of potassium hydroxide (KOH)/potassium methanesulfonate acid (KMSA) eluent using MSA and KOH eluent generators installed in series. This enables analysts to run gradient methods for oligosaccharide separations using an isocratic pump and ensures excellent reproducibility and accuracy in eluent preparation. Our study demonstrates good performance of this technology when applied to the analysis of N-linked oligosaccharides released from human serum IgG and human α-1-acid glycoprotein using a 1 mm high-performance anion-exchange column for oligosaccharide separations. This method delivers superior resolution of uncharged IgG oligosaccharides (glycans) comparable with traditional HPAE-PAD separations using sodium hydroxide/sodium acetate eluents. The neutral glycans are well resolved, including separation of high-mannose species from typical human IgG glycans. Oligosaccharide identification was performed by comparison to known standards in conjunction with selective exoglycosidase digestion of both standards and released glycans. Method precision and accuracy was evaluated by multiple injections. This method simplifies operation and improves the precision and accuracy while requiring no eluent preparation, no sample derivatization, and is orthogonal to CE and HILIC methods.

HPAE-PAD analysis of N-linked oligosaccharides from glycoproteins using dual eluent generation cartridge mode

2019 Spring National Meeting

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