Experimental determination of extinction coefficients by sedimentation velocity analytical ultracentrifugation for accurate quantification of recombinant adeno-associated virus

Recombinant adeno-associated virus (rAAV) is widely used as a gene delivery vector. Sedimentation velocity analytical ultracentrifugation (SV-AUC) is the gold standard for quantifying the ratio of full particles (FPs) to the sum of empty particles (EPs) and FPs (F/E ratio) of rAAVs. Here, we experimentally determined the molar extinction coefficients (ε) and mass extinction coefficients of highly purified FPs and EPs of AAV serotypes 2, 5, 6, 8, and 9 using SV-AUC with interference and multi-wavelength absorbance detection. At 230 nm, the difference in ε between EPs and FPs was the smallest, although the ε of FPs remained 1.2-fold higher than that of EPs. Expectedly, the differences in ε between FPs and EPs were almost identical across serotypes with the same genome length and increased linearly in a genome length-dependent manner, although both sets of ε differed across serotypes. Consequently, accurate quantification of F/E ratio requires the use of distinct ε values for EPs and FPs. The ε per base of single-stranded DNA was independent of serotype and genome length, allowing estimation of the ε of FPs from that of EPs. Coupling these ε values with SV-AUC enables the determination of absolute rAAV concentrations. This study provides practical guidance for accurate absorbance-based rAAV quantification.