Weighing the DNA content of Adeno-Associated Virus vectors with zeptogram precision using nanomechanical resonators

Quantifying the composition of viral vectors used in vaccine development and gene therapy is critical for assessing their functionality. Adeno-Associated Virus (AAV) vectors, which are the most widely used viral vectors for in-vivo gene therapy, are typically characterized using PCR, ELISA, and Analytical Ultracentrifugation which require laborious protocols or hours of turnaround time. Emerging methods such as Charge-Detection Mass Spectroscopy, Static Light Scattering, and Mass Photometry offer turnaround times of minutes for measuring AAV mass, but mostly require purified AAV-based reference materials for calibration. Here, we demonstrate a method for using Suspended Nanomechanical Resonators (SNR) to directly measure both AAV mass and aggregation from a few microliters of sample within minutes. We achieve a resolution near 10 zeptograms which corresponds to 1% of the genome holding capacity of the AAV capsid. Our results show the potential of our method for providing real-time quality control of viral vectors during biomanufacturing.

Collective amplification of nearby nanoparticles in the Coulomb blockade restricted charging of a single nanoparticle

The characterization of charges in oxide supported metal nanoparticles (NP) is of high interest in research fields like heterogeneous catalysis and microelectronics. A general desire is to manipulate the charge of an oxide supported single NP and to characterize afterwards the charge and its interference with the insulating support but also with nearby NPs in the vicinity. By using noncontact AFM (nc-AFM) and Kelvin probe force microscopy (KPFM) in ultra-high vacuum (UHV) and at room temperature we show that a ~5 nm small AuNP can be directly charged with electrons by the AFM tip and that upon the charging, nearby AuNPs sensitively change their electrostatic potential with a large impact on the charge detection by nc-AFM and KPFM. The AuNPs are supported on a 40 nm thick insulating Al2O3 film, which is grown by atomic layer deposition (ALD) on Si(001). Due to Coulomb blockades, the NP charging appears in the form of large and discrete peaks in detuning versus bias voltage curves. Finite element method (FEM) calculations reveal that the large peaks can only be observed when the potentials of nearby insulated NPs get modified by the NP's electron charge, according to the electrostatic induction principle. In view of the number of transferred electrons, we anticipate that after the charging, the electrons are transferred from the AuNP to the NP-Al2O3 interface or into Al2O3 subsurface regions directly underneath.

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.