Evaluating zwitterionic stationary phases for glycan profiling of IgGs from various sources by HPLC-ESI-MS/MS

In this study the separation performance of nine surface grafted zwitterionic chromatography columns for HILIC mode glycan profiling of IgG derived glycans were compared. Glycans were enzymatically released from IgGs of different origin and analysed in the reduced form using HPLC coupled with electrospray ionisation high resolution mass spectrometry analysis, HPLC-ESI-MS/MS. Column comparison was based on the obtained glycan profile including glycan retention, chromatographic peak attributes and obtained resolution. The columns tested included stationary phases with sulfobetaine (SB) and phosphorylcholine (PC) functionality bound to solid core and fully porous particles of different particle and pore size, yielding columns with different pore volumes and surface chemistries. All columns yielded informative glycan profiles with detection and separation of major glycan species expected in the included IgGs, but differences in observed glycan retention and obtained resolution were observed. The surface functionality had the biggest impact on the glycan elution profile with the phosphorylcholine functionality yielding the best overall separation, and especially an improved chromatographic resolution for sialic acid containing versus non-sialylated glycans. Improved chromatographic resolution is especially important when not using mass spectrometry for glycan detection. The different columns yielded different chromatographic profiles of the glycans, and columns with solid core particles generally resulted in shorter analysis time, enabling higher throughput separations which could increase throughput in glycan profiling and quality control.