Deciphering super-heterogeneous glycosylated biotherapeutics by combining hydrophilic interaction...

The structural heterogeneity of glycosylated biotherapeutics, driven by diverse glycan modifications, poses significant challenges for comprehensive characterization and quality assessment of these molecules. Conventional mass spectrometry (MS) approaches often struggle with the complexity of intact glycoprotein analysis due to overlapping glycoform populations. In this study, we present an integrated analytical workflow that combines hydrophilic interaction liquid chromatography (HILIC) with MS detection and a scan-by-scan deconvolution algorithm. HILIC separation efficiently resolves glycoforms based on glycan composition, reducing spectral congestion and enhancing data interpretability. The scan-by-scan deconvolution strategy further simplifies the analysis by independently processing each MS scan, enabling accurate intact mass determination and detailed glycoform profiling. Application of this method to a highly heterogeneous CD24-Fc fusion protein enabled the identification of over 300 distinct glycoforms. This workflow provides a robust and high-resolution platform for intact glycoprotein analysis, supporting biopharmaceutical development, quality control, and structural biology studies.