rapid-glyco-qualitative-assessment-recombinant-proteins-using-fully

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, ...

National Institute of Advanced Industrial Science and Technology

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 glycans considered to be attached to rAAV6 were recognized by  agglutinin (ABA) and that glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was glycosylated with the mucin-type glycans, 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 GlcNAc at T242 in VP3. The mucin-type glycosylated rAAV6 particles were 0.1%-1% of total particles. Further physicochemical and biological analyses revealed that mucin-type glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both  and  compared with rAAV6 without mucin-type glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.

Saho Mizukado

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