Catherine A. Formolo and Karen W. Phinney



Protein-based therapeutics comprise a fast-growing class of pharmaceuticals.  However, the inherent variability of these biologics poses a challenge to the industry.  Protein drugs with the same primary structure can vary greatly in their glycosylation patterns, which in turn can affect many properties of the drug, including safety, efficacy and stability.  Characterizing these glycan structures is therefore an essential step in the evaluation of protein biologics.


For proteins that carry multiple glycosylation sites it is often necessary to not only identify each glycan structure present, but also map each structure to the individual occupied sites.  This can be performed via enzymatic digestion of the protein followed by glycopeptide enrichment and LC-MS/MS analysis.  Here we describe the use of a pentafluorophenylpropyl (F5) stationary phase to resolve sialylated glycopeptides and compare its chromatographic properties with the commonly used C18 phase.  While both phases provided good separation of glycopeptides containing different amino acid sequences we found that resolution of glycopeptides with the same amino acid sequence but differing glycan structures was improved with the F5 phase.  Therefore this stationary phase may be a useful tool for resolving glycopeptide mixtures prior to mass spectrometry analysis.