Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer

We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.

Assessment of genome packaging in AAVs using Orbitrap-based charge detection mass spectrometry

Adeno-associated viruses (AAV) represent important gene therapy vectors with several approved clinical applications and numerous more in clinical trials. Genome packaging is an essential step in the bioprocessing of AAVs and needs to be tightly monitored to ensure the proper delivery of transgenes and the production of effective drugs. Current methods to monitor genome packaging have limited sensitivity, a high demand on labour, and struggle to distinguish between packaging of the intended genome or unwanted side-products. Here we show that Orbitrap based charge detection mass spectrometry allows the ultra-sensitive quantification of all these different AAV bioprocessing products. A protocol is presented that allows the quantification of genome packed AAV preparations in under half an hour, requiring only micro-liter quantities of typical AAV preparations with ~1013 viral genome copies per millilitre. The method quickly assesses the integrity and amount of genome packed AAV particles to support AAV bioprocessing and characterization of this rapidly emerging class of advanced drug therapies.