Site-Specific N-Glycan-Resolved PK Assessment of Therapeutic Proteins Using Hybrid LBA-HRMS and LC-MS/MS

Understanding how glycosylation and its metabolic conversion shape the pharmacokinetics (PK) and pharmacodynamics (PD) of therapeutic proteins is essential for predicting exposure and efficacy. Lot-to-lot comparisons typically rely on aggregate PK metrics that obscure contributions from individual glycoforms, despite their distinct clearance behaviors. Glycan-resolved PK can disentangle these effects by quantifying the in vivo disposition of specific glycoforms and their impact on overall PK/PD. Here, we apply bottom-up LC-HRMS and LC−MS/MS workflows to characterize major site-specific N-glycopeptides and perform glycan-resolved PK analysis for two therapeutic fusion proteins containing multiple glycosylation sites. While ligand-binding assays effectively measure total drug they do not account for the individual glycoforms, the use of both LC-HRMS and LC−MS/MS enabled direct identification and quantitation of individual glycoforms across the dosing interval. Distinct apparent PK profiles were observed among glycoforms within each molecule, and these differences informed their relative contributions to apparent systemic clearance. Together, these results demonstrate the value of glycan-resolved bioanalysis for mechanistic interpretation of PK variability and provide a practical framework for integrating LC-HRMS and LC−MS/MS glycopeptide characterization into the development of complex glycoprotein therapeutics.