Anion exchange chromatography coupled to mass spectrometry for deciphering the complexity of a highly glycosylated fusion protein (“protein HGF”)

Fc-fusion proteins are medicines developed for the treatment of complex diseases which feature enhanced pharmacokinetic properties compared to other therapeutic protein formats. A commonly used strategy for the extension of protein half-life in circulation is the introduction of negative charges on the protein, preferably through N- and O-glycans equipped with negatively charged sialic acid moieties. While enhancing pharmacokinetics, the presence of many glycosylation sites can coincide with a considerable protein heterogeneity, which renders analytical characterisation increasingly difficult. In this work, we demonstrate that the complexity of a highly glycosylated intact Fc-fusion protein can be well resolved using mass spectrometry (MS)-friendly anion exchange chromatography (AEX) with pH gradient elution. The application of the developed method allowed for the separation of 9 clear chromatographic peaks and the acquisition of high-quality protein spectra and thus, MS detection of intact protein isoforms. The resulting data enabled the identification of 268 protein features which represents a ∼ 25-fold increase in detected forms compared to output that could be obtained by simple size exclusion chromatography-mass spectrometry. The underlying cause for the separation selectivity observed in AEX was found to be differential protein sialylation while varying numbers of hexose and N-acetylglucosamine units on glycans were identified as other major contributors to the high heterogeneity observed.