To produce in-house rAAV6, a transgene plasmid (HCRhAAT–hFIX), pAAV-Rep-Cap (AAV 6), and pAd helper were co-transfected into suspended VP) 2.0 cells using the transfection reagent FectoVIR-AAV and cultured in a flask. 4 days after transfection, the cells were harvested and lysed, and the lysate was filtered. rAAV6 particles were purified by affinity chromatography using AAVX prepacked columns. To separate the FPs and EPs, the purified AAVs were processed by cesium chloride ultracentrifugation. FP fractions were collected and dialyzed in 1 × Phospho-buffered saline (PBS) buffer containing 200 mM NaCl and 0.001% poloxamer-188.

Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galβ1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%-1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.