A rapid strategy for domain-specific Fab glycosylation profiling of monoclonal antibodies via selective native deglycosylation and ultrafiltration.

A high-throughput method for domain-specific Fab glycosylation analysis was developed for monoclonal antibodies. Fab glycosylation can affect a product's efficacy and safety. Conventional domain-specific glycan analysis-typically relying on IdeS digestion and affinity chromatography-is labor-intensive. In this study, a mathematical subtraction approach using native Fc and total glycosylation data was initially evaluated but was found to suffer from significant error amplification (up to 8.0%). Accordingly, a practical workflow based on physical separation was established. The method entails rapid enzymatic digestion under native conditions, followed by selective Fc-glycan depletion via ultrafiltration, and subsequent denatured glycan release and analysis using a commercial RapiFluor-MS kit. This workflow drastically reduces sample preparation time from days to hours, rendering it highly suitable for high-throughput process development and in-process control.