Article ; Online: Combined 100 keV Cryo-Electron Microscopy and Image Analysis Methods to Characterize the Wider Adeno-Associated Viral Products.
Journal of pharmaceutical sciences
2024
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 ...
| 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. |
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| Language | English | |||||
| Publishing date | 2024-04-02 | |||||
| Publishing country | United States | |||||
| Document type | Journal Article | |||||
| ZDB-ID | 3151-3 | |||||
| ISSN | 1520-6017 ; 0022-3549 | |||||
| ISSN (online) | 1520-6017 | |||||
| ISSN | 0022-3549 | |||||
| DOI | 10.1016/j.xphs.2024.03.026 | |||||
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| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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