[Development and validation of an anion-exchange high-performance liquid chromatography method for determining the...

To establish and validate an anion-exchange high-performance liquid chromatography (AEX-HPLC) method for the determination of the empty-to-full capsid ratio in recombinant adeno-associated virus (rAAV), and to systematically evaluate its detection accuracy by comparing with the gold standard method of analytical ultracentrifugation (AUC), thereby providing a reliable technical approach for the quality control of rAAV empty-to-full capsid ratio. A UniCore^TM Q strong anion-exchange chromatographic column was used, with 20 mmol/L Bis-Tris propane-acetic acid buffer as the mobile phase A, while the mobile phase B consisted of mobile phase A supplemented with 2.5 mol/L tetramethylammonium chloride. Gradient elution was performed over 17 minutes with a fluorescence detector (λ_ex 280 nm, λ_em 348 nm), and systematic methodological validation of the method was conducted. The method showed good specificity. The spiked recovery rate was >87%, and the relative standard deviations (RSD) of the empty-to-full capsid ratio for repeatability and intermediate precision were 4.91% and 0.99%, respectively, indicating good precision. The method had a good linear relationship within the concentration range of 5.54×10¹¹-4.16×10¹² vg/mL (R²≥0.99). Both the limit of detection and limit of quantitation reached 2.77×10¹⁰ vg/mL, and the sensitivity met the requirements for the detection of low-concentration samples. No significant differences in the results were observed when the pH of the mobile phase fluctuated within the range of 9.2-9.4 or the column temperature varied by ±3 ℃, showing good robustness. Compared with the AUC method, there was no statistically significant difference in the determination results (P=0.156). The AEX-HPLC method established in this study has been fully validated, and all performance indicators meet the requirements for the quality control of rAAV empty-to-full capsid ratio. The method can be directly applied to the detection of the empty-to-full capsid ratio in marketed rAAV drugs. The results obtained by this method are consistent with those obtained by the gold standard AUC method, demonstrating reliable accuracy. By virtue of its charge separation principle, the method possesses unique detection advantages and is simple to operate. Furthermore, it overcomes the limitations of the AUC method, such as high equipment cost, low detection throughput, time-consuming detection, and limited accessibility. It is suitable for the detection of rAAV drug intermediates and the release of final products, and holds significant practical value for promoting the establishment of the quality control system for rAAV drug industrialization.