Abstract
A rigorous analytical assessment of recombinant adeno-associated virus (rAAV)-based drug products is critical for their successful development as clinical candidates. It is especially important to ascertain high purity while simultaneously ensuring low levels of impurities in the final drug product. One approach to evaluate the purity of rAAV drug products is to determine the relative stoichiometry of the three viral proteins (VPs) that comprise an rAAV capsid, and the levels of impurities in the final drug product. Here we present two capillary electrophoresis-western (CE-western) assays for quantifying (1) the relative stoichiometry of VP using the anti-AAV B1 antibody, and (2) residual levels of a baculovirus protein impurity, GP64, using the anti-GP64 antibody. In each assay, various purified samples from diverse AAV serotypes were analyzed to determine their VP ratio or GP64 levels. The ratio of VP3/VP1 in rAAV samples was correlated with biological activity, and the clearance of GP64 from the manufacturing process was demonstrated. The results obtained from both assays were further supported by liquid chromatography-mass spectrometry analyses. Overall, we report that CE-western is a high-throughput platform that utilizes low sample volumes for a rapid, sensitive, and robust assessment of the identity, composition, and purity of rAAV drug products.