Combined 100 keV Cryo-Electron Microscopy and Image Analysis Methods to Characterize the Wider Adeno-Associated Viral Products

Haruka Nishiumi; Kiichi Hirohata; Mitsuko Fukuhara; Aoba Matsushita; Yasuo Tsunaka; Mark Allen Vergara Rocafort; Takahiro Maruno; Tetsuo Torisu; Susumu Uchiyama

doi:10.1016/j.xphs.2024.03.026

Combined 100 keV Cryo-Electron Microscopy and Image Analysis Methods to Characterize the Wider Adeno-Associated Viral Products

J Pharm Sci. 2024 Apr 2:S0022-3549(24)00123-0. doi: 10.1016/j.xphs.2024.03.026. Online ahead of print.

Authors

Haruka Nishiumi¹, Kiichi Hirohata¹, Mitsuko Fukuhara², Aoba Matsushita¹, Yasuo Tsunaka¹, Mark Allen Vergara Rocafort¹, Takahiro Maruno², Tetsuo Torisu¹, Susumu Uchiyama³

Affiliations

¹ Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
² Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; U-medico Inc., 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
³ Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; U-medico Inc., 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: suchi@bio.eng.osaka-u.ac.jp.

PMID: 38570072
DOI: 10.1016/j.xphs.2024.03.026

Abstract

Adeno-associated viruses (AAVs) are effective vectors for gene therapy. However, AAV drug products are inevitably contaminated with empty particles (EP), which lack a genome, owing to limitations of the purification steps. EP contamination can reduce the transduction efficiency and induce immunogenicity. Therefore, it is important to remove EPs and to determine the ratio of full genome-containing AAV particles to empty particles (F/E ratio). However, most of the existing methods fail to reliably evaluate F/E ratios that are greater than 90 %. In this study, we developed two approaches based on the image analysis of cryo-electron micrographs to determine the F/E ratios of various AAV products. Using our developed convolutional neural network (CNN) and morphological analysis, we successfully calculated the F/E ratios of various AAV products and determined the slight differences in the F/E ratios of highly purified AAV products (purity > 95 %). In addition, the F/E ratios calculated by analyzing more than 1000 AAV particles had good correlations with theoretical F/E ratios. Furthermore, the CNN reliably determined the F/E ratio with a smaller number of AAV particles than morphological analysis. Therefore, combining 100 keV cryo-EM with the developed image analysis methods enables the assessment of a wide range of AAV products.

Keywords: Adeno-associated virus (AAV); Analytical ultra-centrifugation; Biopharmaceutical characterization; Gene therapy; Image analysis; Machine learning; Morphology; Neural network(s); Therapeutic drug monitoring; Viral vector(s).