Glycan profiling of therapeutic monoclonal antibodies: Rapid, non-denaturing single-step digestion via pH-tuned enzymatic cleavage

N-glycosylation of monoclonal antibodies (mAbs) critically affects their efficacy, stability, and immunogenicity, necessitating efficient and sensitive glycan analysis. Hydrophilic Interaction Liquid Chromatography with Fluorescence Detection (HILIC-FLD) is widely used for antibody N-glycan profiling and glycosylation heterogeneity control due to its high sensitivity and strong applicability, primarily by cleaving N-glycans from antibodies with PNGase F followed by liquid chromatography or coupled mass spectrometry (MS) analysis. Conventional PNGase F digestion protocols often require protein denaturation before digestion and overnight incubation, which may cause the preparation process cumbersome, time-consuming and toxic. To address these limitations, we developed a rapid, non-denaturing enzymatic cleavage method by selecting an appropriate buffer system and regulating the pH of the reaction system for mAbs. The method requires only a single-step digestion and enhances enzyme access to N-glycans by modulating antibody conformation and surface charge through pH adjustment, enabling efficient cleavage. This method achieves N-glycan release within 20 min—48 times faster than conventional overnight digestion—while eliminating toxic denaturants, delivering superior performance in time efficiency, operational simplicity, safety, and environmental friendliness. Validation with trastuzumab (IgG1) and sintilimab (IgG4) showed nearly identical N-glycan profiles to conventional methods, retaining chromatographic properties and yielding comparable FA2 peak areas and glycoform abundances. MS revealed consistent glycoform profiles between methods (27 vs. 25 glycoforms in optimized vs. conventional), with strong abundance correlation. These results demonstrate that the method is not only applicable to multi-sample scenarios but also fully MS compatible, offering a robust tool for biopharmaceutical development, batch consistency assessments, and biomarker discovery in glycobiology research.