biostudies-literatureYamaguchi YJapan Agency for Medical Research and Development10405-10416https://www.ebi.ac.uk/biostudies/studies/S-EPMC12096344biostudies-literatureUnknown97(19)Recombinant adeno-associated virus (rAAV) has attracted attention as a gene therapy vector. Monitoring the percentage of full particles (FPs) to the sum of empty particles (EPs) and FPs (F/E ratio) is required to optimize the rAAV production conditions; however, there is a lack of analytical methods to identify FPs and EPs and quantify the F/E ratio of rAAV without purification. Here, we established a direct analysis method for identifying FPs and EPs and quantifying the F/E ratio and genomic titer of unpurified rAAV in crude cell lysate and conditioned medium by mass photometry (MP). MP can detect the events of both molecules that bind to the glass surface and molecules that unbind from the glass surface. Few unbinding molecules were detected in the cell lysate and conditioned medium, butAnalytical chemistryDirect Identification and Quantification of Recombinant Adeno-Associated Virus in Crude Cell Lysate and Conditioned Medium by Mass Photometry.PMC12096344JP24se0123004h0101JP18ae0201001Torisu TMatsushita AShimojo SFukuhara MUchiyama STsunaka YYamaguchi YShibuya RBandoh KfalseDirect Identification and Quantification of Recombinant Adeno-Associated Virus in Crude Cell Lysate and Conditioned Medium by Mass Photometry.Recombinant adeno-associated virus (rAAV) has attracted attention as a gene therapy vector. Monitoring the percentage of full particles (FPs) to the sum of empty particles (EPs) and FPs (F/E ratio) is required to optimize the rAAV production conditions; however, there is a lack of analytical methods to identify FPs and EPs and quantify the F/E ratio of rAAV without purification. Here, we established a direct analysis method for identifying FPs and EPs and quantifying the F/E ratio and genomic titer of unpurified rAAV in crude cell lysate and conditioned medium by mass photometry (MP). MP can detect the events of both molecules that bind to the glass surface and molecules that unbind from the glass surface. Few unbinding molecules were detected in the cell lysate and conditioned medium, but2025-01-01T00:00:00Z2025 May2026-06-01T22:21:22.034Z2026-05-22T03:07:50.866ZS-EPMC120963444032617110.1021/acs.analchem.5c00793