Analytical dissection of minor glycoforms and glycoprotein associations in rAAV preparations by multimodal...

Accurate glycan analysis of viral vectors is essential for evaluating pharmaceutical quality. Recent advances in mass spectrometry-based analytical technologies have achieved glycosylation detection in adeno-associated viruses (AAVs). However, because only a minor subpopulation (< 1%) of recombinant AAV (rAAV) particles may carry glycans or associate with glycoproteins, distinguishing genuine AAV glycosylation from that of co-purified glycoproteins remains technically challenging, highlighting the need for analytical strategies that minimize glycan misassignment and reliably identify glycoprotein interactions. Here, we present a multimodal glycoproteomic approach to discriminate rare glycosylation events on rAAV capsids from glycosylated host-derived proteins associated with the particles. We employed an ultrasensitive lectin microarray coupled with a broadly reactive anti-AAV antibody to detect O-glycan-binding lectin signals in several rAAV preparations. Notably, a distinct signal was observed for Urtica dioica agglutinin (UDA). Subsequent liquid chromatography-tandem mass spectrometry, combined with UDA-based dual enrichment at both protein and peptide levels, identified a divalently high-mannose N-glycosylated peptide derived from the host AAV receptor (AAVR). Monovalent high-mannose N-glycopeptides of AAVR and Mac-2 binding protein were additionally detected using single-step protein-level enrichment, indicating an avidity-driven UDA binding mechanism. However, no N-glycosylation was detected on the rAAV capsids themselves. These findings underscore the value of integrated multimodal glycoproteomic workflows for resolving low-abundance glycosylated species and offer new insights into host-derived hitchhiker glycoproteins that may affect rAAV characterization and quality control.