目的:建立分子排阻色谱(SEC)串联紫外检测器(UV)、多角度激光散射检测器(MALS)和示差检测器(RI)的方法分析重组腺相关病毒(rAAV)的颗粒滴度和实心率。方法:对rAAV、rAAV空壳对照品和二者混合样品进行SEC-UV-MALS-RI串联分析,流动相为50 mmol·L-1磷酸盐缓冲液,流速为0.3 mL·min-1,进样量为50 μL,温度为室温,采集时间为30 min;紫外检测器选择260 nm和280 nm双通道;18角度激光散射检测和示差检测采用系统默认设置;用WYATT软件分析结果。结果:样品颗粒滴度为4.816×1012 vp·mL-1,RSD为0.37%;空壳对照品颗粒滴度为3.199×1012 vp·mL-1,RSD为0.75%。按不同预设比例混合的5份样品颗粒滴度的RSD均在3%以内,实心率RSD均在4%以内。混合样品的颗粒滴度和实心率测定结果与预期值基本一致。结论:采用SEC-UV-MALS-RI方法可以分析rAAV制品的颗粒滴度、实心率等项目,适用于rAAV制品颗粒滴度和实心率的质量控制。
Objective: To analyze the particle titer and full rate of a recombinant adeno-associated virus (rAAV) by using size exclusion chromatography (SEC) with tandem ultraviolet detector (UV), multi angle laser scattering detector (MALS) and differential detector (RI). Methods: SEC-UV-MALS-RI series analysis was performed on an rAAV, its empty shell reference substance, and a mixture of the two samples. The mobile phase was 50 mmol·L-1 phosphate buffer, with a flow rate of 0.3 mL·min-1, and a sample loading volume of 50 μL. The temperature is room temperature and the collection time was 30 min. The UV detector selected dual channels of 260 nm and 280 nm. 18 angle laser scattering detection and differential detection adopted the system default settings. The results were analyzed using WYATT software. Results: The particle titer of the sample was 4.816×1012 vp·mL-1, with an RSD of 0.37%. The particle titer of the empty shell reference substance was 3.199×1012 vp·mL-1, with an RSD of 0.75%. The particle titer RSD of the 5 samples mixed according to the preset different ratios were all within 3%, and the RSD of full ratio was all within 4%. The measurement results of particle titer and full ratio of the mixed sample were basically consistent with the expected values. Conclusion: The SEC-UV-MALS-RI method can be used to analyze the particle titer, full ratio, and other items of rAAV products, which is suitable for quality control of particle titer and full ratio of rAAV products.
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