重组腺相关病毒9型空壳率阴离子交换高效液相色谱检测方法的建立及验证  

Development and verification of anion⁃exchange high⁃performance liquid chromatography for determination of empty capsid ratio in recombinant adeno⁃associated virus type 9

作　　者：史新昌[1] 王凤阳 刘亚辉 周勇[1] 刘宾 SHI Xinchang;WANG Fengyang;LIU Yahui;ZHOU Yong;LIU Bin(NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,National Institutes for Food and Drug Control,Beijing 100050,China;不详)

机构地区：[1]中国食品药品检定研究院、国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室、国家药品监督管理局生物制品质量研究与评价重点实验室,北京100050 [2]上海泰昶生物技术有限公司,上海201413 [3]上海天泽云泰生物医药有限公司,上海201203

出　　处：《中国生物制品学杂志》2024年第5期519-526,共8页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2023YFC3403305,2021YFFO600804).

摘　　要：目的建立检测重组腺相关病毒9型(recombinant adeno-associated virus type 9,rAAV9)空壳率的阴离子交换高效液相色谱(anion-exchange high-performance liquid chromatography,AEX-HPLC)法,并进行验证。方法采用基于电荷差异的AEX-HPLC法,通过对流动相洗脱梯度、流动相pH、色谱柱柱温、流速、样品浓度、进样体积和荧光检测器检测波长进行优化,建立检测rAAV9空壳与实心病毒比例的方法,并对方法的专属性、线性、检测限、定量限、精密度和准确度进行验证,确认方法可行性。结果利用CIMac AAV full/empty-0.1 mL分析柱,以20 mmol/L 1,3-二[三(羟甲基)甲氨基]丙烷(BIS-Tris propane,BTP)为流动相A,20 mmol/L BTP+1 mol/LNaCl为流动相B进行梯度洗脱,流动相pH均为9.0,柱温20℃,流速1 mL/min,样品浓度4×10^(12)vg/mL,进样体积10μL,激发波长为280 nm,发射波长为330 nm,可实现rAAV9空壳与实心病毒的基线分离及定量检测。方法验证结果表明,制剂缓冲液无干扰,专属性较好;rAAV9在(1.6~8)×10^(12)vg/mL范围内线性关系良好,r=0.993;检测限为5×10^(10)vg/mL,定量限为1×10^(11) vg/mL;重复性和中间精密度RSD分别为2.95%和2.10%;准确率均不低于80%。结论建立了一种高灵敏度且可快速检测rAAV9空壳与实心病毒比例的AEX-HPLC法,可用于基因治疗产品空壳率的分析及质量控制。Objective To develop and verify an anion-exchange high-performance liquid chromatography(AEX-HPLC)method for the determination of empty capsid ratio of recombinant adeno-associated virus type 9(rAAV9).Methods AEXHPLC based on the differences in surface charge was used to establish a method for detecting the ratio of empty and full capsid rAAV9 by optimizing the elution gradient of mobile phase,pH,column temperature,flow rate,sample concentration,injection volume and detection wavelength of fluorescence detector.The specificity,linearity,limit of detection(LOD),limit of quantitation(LOQ),precision and accuracy of the method were verified to confirm the feasibility.Results Using a CIMac AAV full/empty-0.1 mL column,20 mmol/L BIS-Tris propane(BTP)as mobile phase A and 20 mmol/L BTP+1 mol/L NaCl as mobile phase B,gradient elution was performed with pH of 9.0,column temperature of 20℃,flow rate of 1 mL/min,sample concentration of 4×10^(12)vg/mL,injection volume of 10μL,excitation wavelength of 280 nm and emission wavelength of 330 nm,which realised the baseline isolation and quantitative detection of empty and full capsid rAAV9.The verification results of the method showed that the preparation buffer had no interference with good specificity;rAAV9 showed a good linear relationship in the range of(1.6-8)×10^(12)vg/mL,r=0.993;the LOD was 5×10^(10) vg/mL,and the LOQ was 1×10^(11) vg/mL;the RSD of repeatability and intermediate precision were 2.95%and 2.10%,respectively;the accuracy rates were not less than 80%.Conclusion A highly sensitive and rapid AEX-HPLC method for determination of the ratio of empty capsid to full capsid rAAV9 was developed,which could be used for the analysis of empty capsid rate and quality control in gene therapy products.

关 键 词：重组腺相关病毒 基因治疗 阴离子交换高效液相色谱法 空壳病毒 实心病毒 质量控制 

分 类 号：R917[医药卫生—药物分析学]