NEW! PNGase F for Hydrolysis of N-glycans

November 29, 2021 | New Product, News |

We are happy to announce that PNGase F for in-solution hydrolysis of N-glycans from glycoproteins is added to our portfolio of SmartEnzymes!
 

PNGase F is a glycoamidase hydrolyzing the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high-mannose oligosaccharides. The Fc N-glycan play a crucial role in the effector function of antibodies but may add complexity to protein characterization assays. Removal of N-glycans from glycoproteins is widely used for sample preparation for MS analysis – to reduce the protein heterogeneity and enable released glycan analysis – and to study the functional role of the N-glycans.

 

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.

 

 
RapiFluor-MS™ labelled N-glycans released from trastuzumab (top), etanercept (middle) and RNase B (bottom) 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.


 

 

 

Immobilized PNGase F – For hydrolysis of N-glycans from 0.2 mg glycoproteins under native conditions.

 

PNGase F – Lyophilized enzyme for hydrolysis of N-glycans from 1 mg glycoprotein.