Prolonged Trappingof Adeno-Associated Virus CapsidsReveals that Genome Packaging Affects Single-Ion Mass SpectrometryMeasurements

Recombinant adeno-associated viruses (rAAVs) play an important role in gene therapy, yet the optimal preparation of these biotherapeutics remains challenging, with often incomplete incorporation of genome cargo, negatively affecting therapeutic use. Genome packaging has traditionally been difficult to investigate. Charge detection mass spectrometry (CDMS) has gained prominence, as it provides the ability to mass analyze and resolve empty and filled rAAVs, enabling the quantitative determination of empty-to-filled rAAV ratios. Such measurements require a high mass resolving power and depend on the unbiased detectability of the distinct rAAV particles. The mass resolving power in the Orbitrap mass analyzers scales with transient recording times. Therefore, we extended the capability of recording ions from 1 or 2 to 24 s. When we record these 24 s transients to analyze rAAVs, we not only observe a substantial improvement in accuracy and mass resolution but also find that this can lead to erroneous artifacts in the empty-to-filled ratios. Artifacts originate from the distinct behavior between orbiting ions from either empty or filled particles, mainly due to differences in desolvation and charge losses. Ions from empty rAAVs appear much more susceptible to charge losses, which negatively affect their tracing, artificially decreasing the empty-to-filled ratios. Further elucidating the causes of this undesirable ion behavior, we provide the means to minimize charge losses, thus regaining accurate AAV quantification at an increased charge precision and mass-resolving power. As the Orbitrap-based CDMS has become a method of choice to determine this important quality control attribute, our findings are important to avoid reporting incorrect empty-to-filled ratios.