Rapid characterization of adeno-associated virus (AAV) capsid proteins using microchip ZipChip CE-MS

Abstract

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) was applied for the rapid characterization of AAV intact VPs, specifically full and empty viral capsids of serotypes AAV6, AAV8 and AAV9, which was accomplished using 5 min of analysis time. 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, including truncated VP3 variants most likely generated by leaky scanning as well as unacetylated and un-cleaved VP3 proteoforms. 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. 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.

Keywords: Adeno-associated virus; Characterization; Gene therapy; Microchip CE-MS; Post translation modifications; Product quality monitoring.

Fig. 1

A ZipChip CE-MS total ion electropherograms. The maximum intensity value is listed on the right side of each electropherogram in counts. B Extracted MS spectra intensity of each VP peak when evaluating the use of DMSO in BGE during sample preparation and analysis. Empty AAV8 capsids were used for this analysis and VP peaks were detected in the electropherograms between 3.2 and 3.8 min. Condition 1 (red): no DMSO is used in the BGE for either sample prep or analysis. Condition 2 (blue): no DMSO is used in the BGE during sample prep, but 4% DMSO is added to the BGE during analysis. Condition 3 (purple): 4% DMSO is added to the BGE for both sample prep and analysis

Fig. 2

ZipChip CE-MS Limit of Detection testing for AAV capsid analysis. A Total ion electropherograms of AAV8E VPs analysed at decreasing concentrations. The peaks representing the VP proteins are encased in the grey box. The other peaks detected are those of host cell contaminants. The VP peak area shown is an average of the total VP peak area of the 5 injections run at each concentration of capsids injected. B MS spectra of the total area of the VP peaks in the electropherograms described in (A). Spectral intensity value shown is the average spectral intensity of area for the 5 injections run at each concentration of capsids injected. Injection 3 of each sample concentration analysed is used as a representative injection for the electropherograms and the MS spectra shown in (A) and (B), respectively

Fig. 3

Total ion electropherograms for empty (top) and full (bottom) capsids analysed for serotypes AAV6 (left), AAV8 (middle), and AAV9 (right). Host cell contaminants are detected in empty capsid samples, but not full capsid samples. Injection 3 from the analysis of each sample analysed is used for illustrative purposes

Fig. 4

Total ion electropherograms illustrating the VP proteoforms and VP fragments detected in the serotypes analysed. Injection 3 from the analysis of the full capsids for each serotype is used for illustrative purposes: (top) AAV6; (centre) AAV8; (bottom) AAV9