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<p>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 (&lt; 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 <i>O</i>-glycan-binding lectin signals in several rAAV preparations. Notably, a distinct signal was observed for <i>Urtica dioica</i> 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 <i>N</i>-glycosylated peptide derived from the host AAV receptor (AAVR). Monovalent high-mannose <i>N</i>-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 <i>N</i>-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.</p> Graphical Abstract <p>Estimated <i>Urtica dioica</i> lectin-binding detection. (<b>A</b>) Although recombinant adeno-associated virus (rAAV) does not contain <i>N</i>-glycans, a <i>Urtica dioica</i> agglutinin (UDA) signal is detected on the antibody-overlay lectin microarray, likely due to glycosylated hitchhiker proteins. (<b>B</b>) Peptide-level UDA capture enables efficient detection of a divalent high-mannose glycopeptide derived from the AAV receptor (AAVR) by selectively removing free peptides and low-affinity glycopeptides</p> <p></p>

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

  • 期刊论文

  • 发表日期：

    2025年8月7日

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  • Atsushi Kuno,
  • Hiroaki Sakaue,
  • Sachiko Koizumi,
  • Azusa Tomioka,
  • Saho Mizukado,
  • Yuki Yamaguchi,
  • Mitsuko Fukuhara,
  • Yasuo Tsunaka,
  • Hiroyuki Kaji,
  • Susumu Uchiyama

摘要

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.

Graphical Abstract

Estimated Urtica dioica lectin-binding detection. (A) Although recombinant adeno-associated virus (rAAV) does not contain N-glycans, a Urtica dioica agglutinin (UDA) signal is detected on the antibody-overlay lectin microarray, likely due to glycosylated hitchhiker proteins. (B) Peptide-level UDA capture enables efficient detection of a divalent high-mannose glycopeptide derived from the AAV receptor (AAVR) by selectively removing free peptides and low-affinity glycopeptides