A unified HILIC-FLD-MS workflow for simultaneous peptide and released N-glycan mapping of protein biopharmaceuticals

Quality control of protein biopharmaceuticals has evolved from separate single-attribute assays toward consolidated methods that monitor diverse critical quality attributes in fewer analyses, most often using bottom-up RPLC-MS/MS workflows. Despite its central role in protein characterization, RPLC remains limited in resolving deamidated peptides, retaining hydrophilic species, and separating N-glycoforms, with glycan analysis still often requiring a dedicated HILIC-FLD assay after their release and labeling.

We introduce a unified HILIC-FLD-MS multi-attribute method that brings peptide mapping and released N-glycan profiling into a single chromatographic run. The method exploits distinct HILIC retention windows of tryptic peptides and RapiFluor-MS-labeled glycans, enabling concurrent monitoring of peptide- and glycan-related quality attributes with minimal interference. In this configuration, HILIC retains the strengths of released glycan analysis, including superior glycan resolution and reliable FLD-based quantification, while broadening peptide mapping beyond the limitations of RPLC. Comparative experiments further revealed fewer temperature-induced in-column peptide artifacts under HILIC conditions and demonstrated that chromatographic separation can induce cleavage of terminal sialic acids, especially in glycopeptides.

The proposed method extends HILIC's role from a dedicated glycan separation mode to an integrated, complementary platform for multi-attribute monitoring in biopharmaceutical quality control. By combining peptide-level information with released glycan quantification, the workflow enables more efficient product evaluation while preserving a reliable readout for glycosylation assessment.