Enzymatic Release of N-Glycans from Monoclonal Antibodies Using PNGase F v1

N-glycan influences the efficacy and safety of monoclonal antibodies. (1-3) N-glycan analysis is a critical quality attribute during drug development. This process involves the release of glycans from the peptide backbone. Two popular methods used in glycan analysis are chemical and enzymatic release. The chemical approach is less popular due to the use of highly toxic chemicals and the potential production of unpredictable modifications to the released glycans. (4-6) Most current studies employ enzymatic methods, using Peptide: N-Glycosidase F (PNGase F) as the main enzyme to release N-glycans.(7-9) PNGase F can cleave most N-glycans, including large and complex species, with the only exceptions for non-mammalian glycans containing a 1,3-core fucose. (10) This protocol provides a detailed, step-by-step method for releasing N-glycans from monoclonal antibodies using PNGase F. This workflow is optimized for 1 mg of antibody but can be scaled proportionally.