Comparative characterization of the glycosylation profiles of an influenza hemagglutinin produced in plant and insect hosts.

The main objective of this study was to characterize the N-linked glycosylation profiles of recombinant hemagglutinin (HA) proteins expressed in either insect or plant hosts, and to develop a mass spectrometry based workflow that can be used in quality control to assess batch-to-batch reproducibility for recombinant HA glycosylation. HA is a surface glycoprotein of the influenza virus that plays a key role in viral infectivity and pathogenesis. Characterization of the glycans for plant recombinant HA from the viral strain A/California/04/09 (H1N1) has not yet been reported. In this study, N-linked glycosylation patterns of the recombinant HAs from both insect and plant hosts were characterized by precursor ion scan-driven data-dependent analysis followed by high-resolution MS/MS analysis of the deglycosylated tryptic peptides. Five glycosylation sites (N11, N23, N276, N287, and N481) were identified containing high mannose type glycans in plant-expressed HAs, and complex type glycoforms for the insect-expressed HA. More than 95% site occupancy was observed for all glycosylation sites except N11, which was 60% occupied. Multiple-reaction monitoring based quantitation analysis was developed for each glycopeptide isoform and the quantitative results indicate that the Man(8) GlcNAc(2) is the dominant glycan for all sites in plant-expressed HAs. The relative abundance of the glycoforms at each specific glycosylation site and the relative quantitation for each glycoform among three HAs were determined. Few differences in the glycosylation profiles were detected between the two batches of plant HAs studied, but there were significant differences between the glycosylation patterns in the HAs generated in plant and insect expression hosts.