Comparison of two approaches for quantitative O-linked glycan analysis used in characterization of recombinant proteins.

The principal aim of this study was to demonstrate the optimization and fine-tuning of quantitative and nonselective analysis of O-linked glycans released from therapeutic glycoproteins. Two approaches for quantitative release of O-linked glycans were examined: ammonia-based β-elimination and hydrazinolysis deglycosylation strategies. A significant discrepancy in deglycosylation activity was observed between the ammonia-based and hydrazinolysis procedures. Specifically, the release of O-glycans from glycoproteins was approximately 20 to 30 times more efficient with hydrazine compared with ammonia-based β-elimination reagent. In addition, the ammonia-based reagent demonstrated bias in the release of particular glycan species. A robust quantitative hydrazinolysis procedure was developed for characterization of O-glycans. The method performance parameters were evaluated. It was shown that this procedure is superior for quantitative nonselective release of O-glycans. Identity confirmation and structure elucidation of O-glycans from hydrophilic interaction chromatography (HILIC) fractions was also demonstrated using linear ion trap Fourier transform mass spectrometry (LTQ FT MS) with mass accuracy below 1ppm.