Variation of VP2 stoichiometry and deamidation of VP1 during production and their impacts on the transduction efficiency of AAV vectors

Recombinant adeno-associated viruses (rAAVs) are a leading platform for <i>in vivo</i> gene therapy. However, limited information is available on the fluctuation of quality attributes (QAs), during manufacturing using the HEK293 suspension cell line system, which is suitable for small- to large-scale production using bioreactors. In this study, we evaluated 14 QAs and their variations, including the viral protein (VP) stoichiometric ratio and deamidation ratios of asparagine residues, of the products for AAV8 with enhanced green fluorescent protein as a gene of interest, which were produced employing essentially the same production procedures with slight modification in the upstream conditions. The VP stoichiometric ratios of the manufactured adeno-associated virus (AAV) vectors differed significantly among the products. Correlation analysis of the QAs revealed the attributes that could affect the transduction efficiency. Notably, the VP2 stoichiometric ratio was positively correlated with the transduction efficiency. The deamidation ratios of N57 and N94 in VP1 had a negative tendency to the transduction efficiency. Consistently, the accelerated stability testing showed a negative correlation of the time-dependent increase in the deamidation ratios of the two asparagine residues with the transduction efficiency. This study highlights the importance and implications of the examined QAs in the development of rAAVs.