Spectral Interferences Impede the High-Resolution Mass Analysis of Recombinant Adeno-Associated Viruses

Abstract

Recombinant adeno-associated viruses (rAAVs) are the leading platform for in vivo delivery of gene therapies, with several already approved for clinical use. However, the heterogeneity and structural complexity of these viral particles render them challenging targets to characterize. Orbitrap-based native mass spectrometry (MS) is a method capable of directly characterizing intact megadalton protein assemblies. Here we used such an approach to characterize four different preparations of rAAV8 (two empty and two filled) differing in both their transgene and relative capsid protein isoform (i.e. VP1, VP2 and VP3) content. Interestingly, in native MS measurements of these samples, we observe complicated, unusual, and dramatically different spectral appearances between the four rAAV preparations that cannot be rationalized or interpreted using conventional approaches (i.e. charge state deconvolution). By combining high-resolution native MS, single particle charge detection MS, and spectral simulations, we reveal that these unexpected features result from a combination of stochastic assembly-induced heterogeneity and divergent gas phase charging behaviour between the four rAAV preparations. Our results stress the often-neglected heterogeneity of rAAVs, but also highlight the pitfalls of standard high-resolution mass analysis for such particles. Finally, we show that charge detection MS and spectral simulations can be used to tackle these challenges.

Orbitrap-Based Mass and Charge Analysis of Single Molecules

“…In 2021, we proposed that AAV capsid assembly is stochastic, and native mass spectra are actually representative of multiple overlapping combinations of VP stoichiometries, generating complex spectral interferences. Orbitrap-based CDMS revealed that the spectral appearance of an AAV preparation is closely related to its ESI charging behavior, with lower charged AAVs correlated with more apparent heterogeneity . This suggests that Orbitrap-based CDMS could be an efficient tool to predict native MS distributions of AAVs.…”

“…Orbitrap-based CDMS revealed that the spectral appearance of an AAV preparation is closely related to its ESI charging behavior, with lower charged AAVs correlated with more apparent heterogeneity. 59 This suggests that Orbitrap-based CDMS could be an efficient tool to predict native MS distributions of AAVs. Overall, Orbitrap-based CDMS is particularly well-adapted for a thorough characterization of AAVs, which fall in a size regime where only a few analytical techniques are available.…”

Single-Ion Mass Spectrometry for Heterogeneous and High Molecular Weight Samples

“…There is considerable interest in resolving the subpopulations, because they provide detailed information on the distribution of VP1, VP2, and VP3. Heck and co-workers have reported several attempts to resolve them using conventional MS. − While they were able to resolve sharp peaks in the m / z spectrum, a complete analysis could not be performed because of complexity. Each peak in the mass distribution in Figure b spawns more than ten peaks in the m / z spectrum due to different charge states.…”

Antibody Binding to Recombinant Adeno Associated Virus Monitored by Charge Detection Mass Spectrometry

“…The result is a capsid with an average mass of ∼3.75 MDa with a distribution ∼125 kDa wide. Such a heterogeneous mass distribution cannot be determined using conventional MS, but it can be measured by charge detection mass spectrometry (CD-MS) . CD-MS has previously been used to examine AAV vector heterogeneity and genome integrity. − In this study, we have used CD-MS to investigate antibody binding to AAV capsids.…”