[Zurück]

S. Zoratto, V. Weiss, G. Friedbacher, C. Buengener, A. Foettinger-Vacha, R. Pletzenauer, M. Graninger, G. Allmaier:
"Nano electrospray differential mobility analysis and molecular weight determination of aerosolized adeno-associated viruses";
Vortrag: European Aerosol Conference EAC2021, University of Leeds, UK; 30.08.2021 - 03.09.2021; in: "EAC2021-Book-of-Abstracts.pdf", (2021).

Adeno-associated virus (AAV) based virus-like particles (VLPs) are popular and high-impact delivery systems in the biopharmaceutical field of gene therapy. To ensure the safety of these bionanoparticles, characterization of VLPs is a mandatory step in product development. In this context, purified AAV , either empty (i.e. containing no genome) or filled (i.e. containing genome), have been analyzed via nano electrospray-differential mobility analysis (nES-DMA), aka gas-phase electrophoretic mobility molecular analysis (nES GEMMA) (Kaufman et al., 1996). nES GEMMA allows surface-dry particle size (electrophoretic mobility diameter, EMD) determination of aerosolized nanoparticles in the single- to at least double-digit nanometer range with particle-number detection at ambient pressure. The VLP´s EMD values can be translated in molecular weight (MW) thanks to EMD/MW correlations based on mainly mass spectrometric data available in literature (Weiss et al., 2015 and 2019).
nES GEMMA analysis yield detection of bio-nanoparticles at 25.1 ± 0.2 nm and 25.9 ± 0.1 nm EMD for empty and filled VLPs, respectively (Figure 1). Diameters correlate well with theoretical value of AAV particles. To ensure repeatability, samples were analyzed at least in triplicate with approx. 5000 nanoparticles for good statistics. Based on the obtained EMDs, the MW of the AAV bionanoparticles were determined to be 3670 ± 69 kDa and 4751 ± 47 kDa, respectively. These values highly correlate with theoretical ones: 3746 kDa and 5076 kDa for empty and filled, respectively.
Moreover, different VLP´s oligomeric states can be generated after treating the samples with heat and shear stress. These oligomeric states have been separated, enriched, and collected via asymmetric field flow fractionation (AFFF), for subsequent analysis with nES GEMMA and atomic force microscopy (AFM).
Both techniques confirm the presence of dimers, trimers, and putative higher VLP oligomers. AFM statistical analysis based on more than 500 nanoparticles resulted in average diameters 30.7 ± 2.4 nm and 25.8 ± 2.4 nm for empty and filled AAV VLPs, respectively (Table 1).
In summary, nES GEMMA proved to be suitable for the analysis of AAV VLP bionanoparticles in the MDa range, and is therefore a promising device for bionanoparticles research, especially in QC analyses and during process development, especially due to its low running cost and sample usage, and good statistic.

Gas-phase electrophoresis, nES GEMMA, AAV, virus-like particle, VLP