Establishment and verification of an imaged capillary isoelectric focusing method for detection of charge heterogeneity of recombinant adeno-associated virus capsid proteins
10.13200/j.cnki.cjb.004282
- VernacularTitle:重组腺相关病毒衣壳蛋白电荷异质性成像毛细管等电聚焦电泳检测方法的建立及验证
- Author:
QIN Xi
- Publication Type:Journal Article
- Keywords:
Imaged capillary isoelectric focusing(iCIEF);
Recombinant adeno-associated virus(rAAV);
Capsid proteins;
Charge heterogeneity
- From:
Chinese Journal of Biologicals
2024;37(8):904-910
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish and verify an imaged capillary isoelectric focusing(iCIEF)method for analyzing the charge heterogeneity of recombinant adeno-associated virus(rAAV)capsid proteins,in order to provide a reliable detection method for characterization research,quality control and process stability monitoring of rAAV products. Methods After denaturation,the sample was analyzed by iCIEF using UV fluorescence detection imaging mode,with injection time of 55 s,voltage of 1 500 V,focusing for 1 min,and then voltage adjusted to 3 000 V for another 10 min. The excitation wavelength was set at 280 nm,and the exposure time of emission fluorescence detection was 80 s. Taking rAAV 5 empty particles(rAAV-E)as the research object,the specificity,precision,linear range,accuracy and limit of quantitation(LOQ)of the method were verified. The established method was used to detect the charge heterogeneity of rAAV 5 full particles(rAAV-F).Results The rAAV-E sample showed two main peaks(Peak1 and Peak2)between pH 6. 9 and pH 7. 1,and no virus capsid protein peak was detected in the blank control. In the six repeated detections,the average pI values of the two main peaks Peak1 and Peak2 of rAAV-E sample were 6. 91 and 7. 04 respectively with both RSDs of 0. 14%,and the average proportions of peak area were 35. 6% and 55. 8%,with RSDs of 1. 1% and 0. 4%,respectively. The RSDs of peak area proportions of peak 1 and peak 2 of rAAV-E sample at three time points was 3. 45% and 3. 38%,and the RSDs of pI were 0. 10%and 0. 11%,respectively. In the range of(4. 08-6. 12)× 1012vp/mL,the rAAV-E concentration showed a good linear relationship with the average peak area of the main peaks,and the linear regression equation was y = 54 888 x-76 556,R~2 = 0. 977. The recovery rates of the the main peak content in rAAV-E samples at 80%,100% and 120% expected concentrations were all in the range of 80%-120%. The LOQ was 1. 02 × 10~(12)vp/mL for Peak 1,and 0. 76 × 10~(12)vp/mL for Peak 2. In addition to Peak 1 and Peak 2,obvious extra peaks were observed in the acidic region(pI < 5. 85)of rAAV-F sample,which was the main characteristic of rAAV-F compared with rAAV-E,and also indicated that rAAV-F had complex charge heterogeneity distribution. Conclusion The established iCIEF method has good specificity,precision and accuracy,and can be used to analyze the charge heterogeneity of rAAV capsid proteins.
