阴离子高效液相色谱法测定重组腺相关病毒空壳率的方法建立与验证

Development and validation of an anion-exchange high-performance liquid chromatography method for determining the empty-to-full capsid ratio in recombinant adeno-associated virus

摘要:

为了建立并验证重组腺相关病毒(recombinant adeno-associated virus, rAAV)空壳率检测的阴离子交换高效液相色谱(anion-exchange high-performance liquid chromatography, AEX-HPLC)法，通过与分析超速离心(analytical ultracentrifugation, AUC)金标准法对比，系统评估其检测准确性，为rAAV空壳率质控提供可靠技术手段，本研究采用UniCore^TM Q强阴离子交换色谱柱，以20 mmol/L双-Tris丙烷-乙酸缓冲液为流动相A，含2.5 mol/L四甲基氯化铵的流动相A为流动相B；梯度洗脱17 min，荧光检测器检测(λ?? 280 nm, λ?? 348 nm)，并对该方法开展系统的方法学验证。结果表明，方法专属性良好；加样回收率>87%，重复性与中间精密度的空壳率相对标准偏差(relative standard deviation, RSD)分别为4.91%和0.99%，精密度良好；在5.54×10¹¹-4.16×10¹² vg/mL浓度范围内线性良好(R²≥0.99)；检测限与定量限均达2.77×10¹⁰ vg/mL，灵敏度满足低浓度样品检测需求；在流动相pH 9.2-9.4、柱温±3 ℃波动时结果无显著差异，耐用性良好；与AUC法对比，测定结果无统计学显著差异(P=0.156)。本研究建立的AEX-HPLC法经全面验证，各项性能指标均符合rAAV空壳率质控要求，可直接应用于市售rAAV药物空壳率检测。该方法与AUC金标准法结果一致，准确性可靠，依托电荷分离原理具备独特检测优势，且操作简便，同时弥补了AUC法设备价格高、检测通量低、检测耗时、普及率不足的缺陷，适合rAAV药物中间产品检测和终产品放行，对推动rAAV药物产业化质控体系建设具有重要实践价值。

Abstract:

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?? 280 nm, λ?? 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.