Acquity uplc beh glycan column

N-glycan analysis of rhIgG1 Fc derived from ALB::hIgG1 Fc chicken was performed by UPLC/MS-MS. Briefly, purified rhIgG1 Fc was incubated with PNGase F for 16 h at 37 °C. Deglycosyated rhIgG1 Fc was precipitated using ethanol and centrifuged at 10,000 g for 10 min. The supernatant containing released N-glycan was transferred to a new tube and dried completely using Speed-Vac concentrator. The dried sample was labelled with procainamide for fluorescence analysis. The labeled N-glycan sample was analyzed and quantified using UPLC/MS-MS. An ACQUITY UPLC BEH Glycan column (1.2 × 150 mm, 1.7 μm; Waters, New Castle, DE) with a fluorescence detector (Waters iClass UPLC) was used for the separation and detection of N-glycans. The LC conditions were as follows: flow rate (0.5 mL/min), column temperature (60 °C), mobile phase buffer A (100 mM ammonium formate, pH 4.5), buffer B (100% acetonitrile), injection volume (8 mL), linear gradient (75–60% B for 46.5 min, 60–0% B for 1.5 min, 0% B for 1 min, 0–75% B for 1 min, and 75% B for 13 min). A high-resolution mass spectrometry, triple-TOF MS (AB SCIEX, Concord, Ontario, Canada), was used for N-glycan identification. The N-glycan distribution was analyzed with Empower (Waters).

Glycan release and derivatization were performed according to Mittermayr et al. [15 (link)]. Chromatographic separation was carried out using ACQUITY UPLC H-Class Bio System with a linear gradient from 22 to 50% acetonitrile using 100 mM ammonium formate solution at pH 4.50 (mobile phase A). Fluorescence detection was set at 250 nm and 420 nm excitation wavelength using 150 × 2.1 mm, 1.7 μm ACQUITY UPLC BEH glycan column coupled to 1.7 μm VanGuard BEH glycan precolumn from Waters Corp. (Milford, MA).

The AB- or ProA-labeled O-glycans were subjected to LC on an Ultimate 3000 LC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ACQUITY UPLC BEH-Glycan column (1.7 μm, 2.1 × 150 mm; Waters). A Q Exactive Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific) was operated in ESI-positive mode. The mobile phase and separation conditions were the same as those used in UPLC. The O-glycans were identified by the elution time of the UPLC chromatogram and the presence of the characteristic oxonium ions of O-glycans in the MS spectrum. The quantity (%) of each glycan relative to total O-glycans (as 100%) was determined from the extracted ion chromatogram (EIC) areas for all observed charge states in LC–ESI–HCD–MS/MS.

Glycan release and derivatization were performed as previously described [20 (link)]. Chromatographic separation was carried out using an ACQUITY UPLC H-Class Bio System with a linear gradient from 22 to 50% of acetonitrile using 100 mM ammonium formate aqueous solution at pH 4.50 as mobile phase A. Fluorescence detection was set at an excitation wavelength of 250 nm and 420 nm for emission, using a 150 × 2.1 mm, 1.7 μm ACQUITY UPLC BEH glycan column coupled with a 1.7 μm VanGuard BEH Glycan Precolumn from Waters Corp. (Milford, MA).