Rapid Characterization of Adeno-Associated Virus (AAV) Capsid Proteins using Microchip ZipChip CE-MS

Adeno-associated viruses (AAVs) are viral vectors used as delivery systems for gene therapies. Intact protein characterization of AAV viral capsid proteins (VPs) and their post-translational modifications is critical to ensuring product quality. In this study, microchip based ZipChip capillary electrophoresis-mass spectrometry (CE-MS) is applied to the rapid characterization of AAV intact VPs, specifically full and empty viral capsids of serotypes AAV6, AAV8 and AAV9. Low levels of dimethyl sulfoxide (4%) in the background electrolyte (BGE) improved MS signal quality and component detection. A sensitivity evaluation revealed consistent detection of VP proteoforms when as little as 2.64 × 106 viral particles (≈26.4 picograms) were injected. Besides the traditional VP proteoforms used for serotype identification, multiple VP3 variants were detected. In AAV6 and AAV8, a VP3 variant where translation starts at the M211 or M212 codon respectively, instead of the expected M203 codon, was identified, most likely generated by leaky scanning. In AAV8 and AAV9 respectively, unacetylated and un-cleaved VP3 proteoforms were observed. Phosphorylation, known to impact AAV transduction efficiency, was also seen in all serotypes analysed. Additionally, low abundant fragments originating from either N- or C-terminus truncation were detected. These fragments are potentially degradation products caused by the v-cath protease, Sf9 cellular response to baculovirus infection, or forced cleavage at a DP sequence through hydrolysis, due to the acidic conditions of the BGE. As the aforementioned VP components can impact product quality and efficacy, the ZipChip’s ability to rapidly characterize them illustrates its strength in monitoring product quality during AAV production.