Acquity uplc glycan beh amide column

The measurements were performed on an Agilent 1290 Infinity II LC System with a binary pump (Agilent Technologies, Inc., Waldbronn, Germany) coupled with maXis™ Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The glycopeptide separation was tested in three different HILIC columns: HALO^® penta-HILIC (2.1 × 150 mm; 2.7 µm; Advanced Materials Technology, Wilmington, Delaware, USA), ACQUITY UPLC Glycan BEH Amide Column (2.1 × 150 mm; 1.7 µm; Waters Corporation, Milford, MA, USA) and SeQuant ZIC-HILIC (2.1 × 150 mm; 3.5 µm; Merck, Darmstadt, Germany). The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient program was optimized to enhance, as much as possible, the resolution of all glycopeptides analyzed in this study. In the case of HALO^® penta-HILIC and ACQUITY UPLC Glycan BEH Amide, the following gradient ((min)/% B) was used: 0/80, 10/80, 25/65, 35/40, 40/40, 43/80, 55/80. For the SeQuant ZIC-HILIC column, a shallower gradient program was used: 0/80, 10/80, 35/65, 45/40, 55/40, 58/40, 70/80. The flow rate was 0.3 mL min⁻¹ for every column, and the column temperature was maintained at 40 °C (if not stated otherwise).

ProA-labeled N-glycans were analyzed using the Waters ACQUITY UPLC H-class system equipped with a Waters ACQUITY UPLC fluorescence detector. Hydrophilic interaction LC-based separation was performed using an ACQUITY UPLC Glycan BEH amide column (1.7 μm, 2.1 × 150 mm; Waters, Milford, MA, USA). Solvents A and B were 50 mM ammonium formate (pH 4.4) and 100% acetonitrile, respectively. The gradient conditions allowed the linear changes of solvent B from 75% to 50% at a flow rate of 0.5 mL/min for 46.5 min. The excitation and emission wavelengths were set at 310 nm and 370 nm, respectively.

Glycan profiling was performed as described previously^{48 (link), 49 (link)}. Briefly, the glycans released from 25 μl of the antibody were labeled and enriched sing a Rapi-Fluor labeling kit, and the resulting products were analyzed on a Waters ACQUITY I class system with an ACQUITY UPLC Glycan BEH amide column (2.1 mm × 150 mm, 1.7 μm particle size). Separation was performed using eluent A (50 mM ammonium formate, pH 4.5) and eluent B (acetonitrile) with a 35-min linear gradient from 25 to 46% of eluent B at a flow rate of 0.4 ml/min. The elent was detected by excitation and emission at 264 nm and 425 nm, respectively.

Ten microlitres (10 μL) of reconstituted released N-glycans were injected into an ACQUITY H-Class UPLC (Waters Corporation, United States) coupled to a Xevo G2S QTof mass spectrometer (Waters Corporation, United States). Samples were separated using an ACQUITY UPLC Glycan BEH amide column (130 Å, 1.7 μm, 2.1 mm × 150 mm, Waters Corporation, United States) at 60°C and 400 μL/min, with a 40 min gradient from 25 to 49% of 50 mM Ammonium Formate (mobile phase A). 100% ACN was used as mobile phase B. RFMS-labelled glycans were excited at 265 nm and measured at 425 nm with an ACQUITY UPLC FLR detector (Waters Corporation, United States). The MS1 profile scans of m/z 400–2,000 were acquired using the Xevo G2S-QTof in positive mode with an acquisition rate of 1 Hz. The electrospray ionisation capillary voltage was set at 2.75 kV, cone voltage at 15 V, desolvation gas flow at 800 L/h, ion source temperature and desolvation temperature were kept at 120°C and 300°C, respectively. Glu1-fibrinopeptide B (Waters Corporation, United States) was used as the LockSpray compound for real-time mass correction. RapiFluor-MS Dextran Calibration ladder (Waters Corporation, United States) was also injected into LC-MS to calibrate the retention time of sample peaks. The retention times were normalised using the dextran calibration curve to Glucose Units (GU).

The purified glycans from the above step were separated on a Waters ACQUITY UPLC system^® using Waters ACQUITY UPLC Glycan BEH Amide column (2.1 × 150 mm, 1.7 mm particle size, 130 Å pore size). A 50 mM ammonium formate solution (pH = 4.4) was used as mobile phase A and a 100% ACN solution was used as mobile phase B. Other parameters for chromatographic separation of glycans included column temperature: 60°C and 75–54% gradient of mobile phase B over 35 min at a flow rate of 0.4 ml/min. The separated glycans were detected using a fluorescence (FLR) detector with an excitation wavelength of 265 nm and an emission wavelength of 425 nm. MS data were recorded on a Bioaccord^® Waters MS system operated in positive ion mode. MS parameters included: m/z range: 50–2000, capillary voltage: 1.5 kV, cone voltage: 45 V. The MS was calibrated using ACQUITY RDa calibrant and wash solution (Cat. 186009012, Waters) and lock mass calibration was performed using ACQUITY RDa lock mass solution (Cat. No. 186009012, Waters).