Glycan analysis of viral vectors is important from the viewpoint of quality evaluation of pharmaceuticals. Due to recent advances in analytical technology, glycosylation of several adeno-associated viruses (AAVs) has been reported. However, the obtained glycosylation pattern and structure are varied and still controversial, suggesting that data acquisition and interpretation using a standardized analytical method are required [1]. In this study, we developed an in-depth analytical method for glycosylation of several serotypes of AAVs using multimodal glycan omics technologies [2]. In brief, 25 recombinant AAVs (rAAVs) among 6 different serotypes were initially subjected to an ultrasensitive lectin microarray we recently improved [3]. Significant signals on O-glycan binding lectins were observed only in rAAV6. The subsequential liquid chromatography-tandem mass spectrometry (LC-MS/MS), combined with the pre-enrichment step using an O-glycan binding lectin and with the post-MS step for glycopeptide identification using a software GRable [4], enabled us to find the mucin-type glycans on viral protein 2 of rAAV6. Interestingly, the glycosylated rAAV6 had a much lower VP1 to VP2/VP3 ratio than non-glycosylated rAAV6. The difference in component proportion leads to the reduced transduction efficiency of rAAV6 [5].

This study was supported by a grant-in-aid from the 'Research and development of core technologies for gene and cell therapy' supported by the Japan Agency for Medical Research and Development (AMED) [grant number JP18ae0201001 and JP18ae0201002].

References

[1] Rumachik N.G., et al., Mol. Ther. Methods Clin. Dev., 18, 98-118 (2020).

[2] Hiono T., et al., J. Proteome Res., 23, 1408-19 (2024).

[3] Boottanun P., et al., Anal. Bioanal. Chem., 415, 6975-84 (2023).

[4] Nagai-Okatani C., et al., bioRxiv (2024). doi:10.1101/2023.10.30.564073.

[5] Yamaguchi Y., et al., Mol. Ther. Methods Clin. Dev., 32, 101256 (2024).