Hydrolysis of N-glycans under Native Conditions using PNGase F

To demonstrate the efficient removal of Fc N-glycans by PNGase F Immobilized and PNGase F Lyophilized under native reaction conditions, the therapeutic antibody trastuzumab was first digested using FabRICATOR. The scFc, LC and Fd’ subunits were then reduced before Fc N-glycan removal using the different formats of PNGase F for 1 h at 37°C. The observed mass shift demonstrates successful removal of the Fc N-glycans.

We can observe a large PNGase F peak eluting just before 7 minutes in the sample deglycosylated using PNGase F Lyophilized. A peak such as this could potentially mask a critically important protein variant. This peak is minimized in the sample digested with PNGase F Immobilized, highlighting a benefit of this format to reduce the amount of enzyme interfering in the final sample.

When using either PNGase F Lyophilized or Immobilized, we can observe in the mass spectra that complete deglycosylation has occurred leaving behind only the protein component. This allows the deglycosylated mass of the protein portion of the scFc to be determined an also allows more confident characterization of any protein modifications that might be present, which would not be as easy to do, if at all, when the glycans are present due to the increased sample heterogeneity.