The influence of pH for the analysis of Adeno-associated virus by nES GEMMA

Abstract

Introduction Adeno-associated virus (AAV) based virus-like particles (VLPs) are popular and high-impact delivery systems. Comprehensive characterization of these particles is an essential yet challenging task. In this context, we investigated the behaviour of AAV VLPs when exposed to electrolyte solutions prepared at different pH levels by nano electrospray differential mobility analysis (nES-DMA, aka gas-phase electrophoretic mobility molecular analysis (nES GEMMA). nES-DMA allows surface-dry particle size (electrophoretic mobility diameter, EMD) determination in the single- to double-digit nanometer range with particle-number detection at ambient pressure. Additionally, we employed atomic force microscopy (AFM) and cryogenic transmission microscopy (CryoTEM) as orthogonal validation techniques to corroborate our findings from nES-DMA. Here, we present challenges and results of our findings.

Methods Purified, empty (not carrying genomic information), and filled (carrying genomic information other than viral) AAV bionanoparticles were obtained from Takeda (Vienna, Austria). The sample buffer was exchanged for ammonium acetate at different pH levels by means of centrifugal filters. EMD determination was carried out via a nES-DMA (TSI Inc, Shoreview, MN, USA) in the size range from 1.95 to 64.9 nm EMD. AFM images were acquired in tapping, constant amplitude mode in air at a scanning rate of 1.99 Hz, over scan areas of 1 and 5 µm2. CryoTEM images were acquired on a 200 kV Thermo Fisher Scientific Glacios instrument at 150k fold magnification.

Results In this study, we demonstrated nES GEMMA's feasibility in analyzing AAV-based VLP preparations with nES GEMMA-compatible electrolyte solution adjusted to different pHs. The two preparations show remarkable differences in terms of EMD and detection efficiency. For the latter, significant VLP depletion is observable solely for the AAV VLPs carrying genomic information at acidic conditions. AFM images of the specified sample reveal a considerable degree of particle aggregation. Lastly, to observe the VLP preparations in their hydrated native state, CryoTEM analysis was performed. Although CryoTEM images do not show particle aggregation, significant structural differences between the samples have been observed.

Innovative aspects • nES GEMMA analysis of AAV VLPs at different pH levels • CryoTEM image reconstruction and particle diameter determination • AAV VLP shows pH-dependent behavior with all employed analytical techniques

Acknowledgements We thank Takeda for providing the AAV samples and Prof. Thomas Heuser for the access to the CryoTEM instrumentation and his support during the corresponding analysis.