Released Glycan Analysis using PNGase F

Application

Comprehensive N-glycan profiling using released glycan workflow, supporting sensitive detection and monitoring of critical quality attributes.

The N-glycosylation profile of therapeutic proteins is a critical quality attribute. It affects both safety and efficacy of the biopharmaceutical and therefore needs to be characterized and monitored during development and production. A common glycan analysis workflow is to release the N-glycans with PNGase F and then label the generated glycans with a fluorescent label such as 2-AB, 2-AA, procainamide or RapiFluor-MS™ (Waters Corporation). The labelled glycans are then separated using HILIC-HPLC or capillary electrophoresis to characterize and quantify the different glycan structures. 

Here, the N-glycans of the therapeutic antibody trastuzumab, the Fc-fusion protein etanercept and the glycoprotein RNase B were analyzed using a released glycan approach. Trastuzumab and etanercept were deglycosylated with PNGase F under native conditions for 1 h at 37°C, while RNase B required denaturation and reduction to be deglycosylated completely. RNase B was therefore treated with RapiGest™ SF surfactant (Waters Corporation) and TCEP before deglycosylation with PNGase F for 10 min at 50°C. The resulting released glycans were labelled with RapiFluor-MS and analyzed by HILIC UPLC-FLD-MS.

PNGase F hydrolysis of N-linked glycans for released glycan analysis

Released glycan analysis allows increased confidence in the identification of low abundant glycan species, making this the method of choice for the comprehensive glycan profiling. This allows any differences in glycosylation to be effectively monitored as some of these glycan types could be determined to be critical quality attributes. For this, efficient deglycosylation is required, and PNGase F has been shown here to be applicable for use on a range of different protein substrates.

Comprehensive glycan profiling to characterize and monitor glycan CQAs

RapiFluor-MS™ labelled N-glycans released from an range of glycoproteins using PNGase F. N-glycans from trastuzumab (top), etanercept (middle) and RNase B (bottom) were hydrolysed using PNGase F before being labelled using RapiFluor-MS™(Waters Corporaion). Labelled glycans were analyzed by HILIC-FLD-MS using a Waters™ BioAccord™ system equipped with a Waters™ ACQUITY Premier Glycan BEH Amide column (130 Å, 2.1 x 150 mm). The fluorescence chromatograms are shown and the glycan structures were annotated using a combination of glucose unit (GU) library search and MS confirmation.

Related Products

Resources