Zwitterionic-type hydrophilic interaction nano-liquid chromatography of complex and high mannose glycans coupled with electrospray ionisation high resolution time of flight mass spectrometry.

In this study we describe a new method for rapid and sensitive analysis of reduced high mannose and complex glycans using zwitterionic-type hydrophilic interaction nano-liquid chromatography (nano ZIC-HILIC, 75 μm I.D.×150 mm) coupled with high resolution nanoelectrospray ionisation time of flight mass spectrometry (nano ESI-TOF-MS). The retention of neutral glycans increases with increasing molecular weight and is higher for high mannose glycans than for complex-type glycans. The selectivity of ZIC-HILIC for sialylated glycans differs from that for the neutral glycans and is believed to involve electrostatic repulsion; therefore, charged glycans are eluted earlier than neutral glycans with comparable molecular weight. Due to the improved sensitivity achieved by employing a ZIC-HILIC nano-column, a range of less common complex glycans has been studied and the high resolution mass spectrometry enabled confirmation of glycan composition for the proposed structures. Good sensitivity for glycans was achieved without prior fluorescent labelling, and the time of the analysis was significantly reduced compared to the separation of glycans on a conventional-size column. The proposed method offers a fast and sensitive approach for glycan profiling applied to analysis of biopharmaceuticals.