Gas-phase electrophoresis (nES GEMMA instrumentation) of adeno-associated viruses S. Zoratto1, T. Heuser2, G. Friedbacher1, R. Pletzenauer3, M. Graninger3, M. Marchetti-Deschmann1, and V.U. Weiss1 1Institute of Chemical Technologies and Analytics, TU Wien, Vienna, 1060, Austria 2Electron Microscopy Facility, Vienna BioCenter Core Facilities GmbH, Vienna, 1030, Austria 3Pharmaceutical Sciences, Baxalta Innovations GmbH (part of Takeda), Vienna, 1220, Austria Keywords: nES GEMMA, DMA, AAV, VLP, gas-phase electrophoresis. Associated conference topics: Special session, 3.4, 4.3 Presenting author email: victor.weiss@tuwien.ac.at In 1996, Kaufman et al. introduced electrophoresis in the gas-phase on a nano Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) aka nES Differential Mobility Analyzer (nES DMA) for globular proteins. (Kaufman, 1996) With such an instrument, analytes are electrosprayed from a volatile electrolyte solution followed by drying of droplets and charge equilibration in a bipolar atmosphere induced e.g. by a radioactive source. Lately, also an alternating bipolar corona discharge was applied for this process. (Weiss, 2020) Single-charged, aerosolized and surface-dry analytes are separated according to their size in a DMA. The obtained monodisperse particles are then counted in a condensation particle counter yielding a spectrum relating a particles surface dry diameter (electrophoretic mobility, EM, diameter) to particle count values. Subsequently, this technique was applied also to other (bio-)nanoparticles, i.a. viruses and virus-like particles (VLPs). (Bacher, 2001 and Weiss, 2019). Via a nES GEMMA setup, information on (bio-)nanoparticle size and size distribution as well as purity of analyte batches can be obtained. Furthermore, by application of a corresponding EM diameter / molecular weight correlation, the molecular weight of an analyte can be assessed from gas-phase electrophoresis data. Turning our attention to VLP based delivery platforms, we investigated gas-phase electrophoresis of an adeno-associated virus (Zoratto, 2021a). Indeed we were able to obtain corresponding spectra. However, we found conditions of the nES crucial for data quality. Presence of VLPs in samples was affirmed by atomic force microscopy (AFM). Figure 1. nES GEMMA spectrum of AAV VLPs (red line) and a corresponding blank (black line) – left. Presence of VLPs was confirmed via AFM measurements – right. (from Zoratto, 2021a) Subsequently, the molecular weight of AAV VLPs was assessed based on gas-phase electrophoresis data. We found these obtained values in excellent accordance with data derived from mass spectrometry. (Zoratto, 2021b) Table 1. Molecular weight values of AAV VLPs obtained via indicated techniques (Zoratto, 2021b). Molecular weight [kDa] Empty VLPs Filled VLPs Theoretical value 3746 5076 nES GEMMA 3670 4751 Native MS 3710 5005 To conclude, gas-phase electrophoresis is capable to yield valuable information in the field of (bio-)nanoparticle characterization – from particle size to VLP molecular weight values as exemplified via AAV based VLPs. We dedicate this presentation to the memory of Prof. Günter Allmaier, TU Wien, Vienna, Austria. Bacher G, Szymanski WW, Kaufman SL et al. (2001) J Mass Spectrom 36, 1038-1052 Kaufman SL, Skogen JW, Dorman FD et al. (1996) Anal Chem 68, 1895-1904 Weiss VU, Pogan R, Zoratto S et al. (2019) Anal Bioanal Chem 411, 5951-5962 Weiss VU, Frank J, Piplits K et al. (2020) Anal Chem 92, 8665-8669 Zoratto S, Weiss VU, Friedbacher G et al. (2021) ACS Omega 6, 16428-16437 Zoratto S, Weiss VU, van der Horst J et al. (2021) J Mass Spectrom 56, e4786