目的:建立毛细管凝胶电泳串联激光诱导荧光检测器(CGE-LIF)方法分析重组腺相关病毒(rAAV)衣壳蛋白VP1、VP2和VP3比例含量并进行方法学验证。方法:CGE-LIF分析rAAV衣壳蛋白方法为:rAAV 样品加入4% SDS-150 mmol·L-1 NEM的溶液预变性(70 ℃,5 min);再加入P503染料标记(70 ℃,10 min);再加入1%SDS进行变性(70 ℃,5 min),然后混匀上机检测。方法验证包括准确性、精密度、线性、检测限、定量限,耐用性等。另外考察了P503、TAMRA、FQ 3种染料条件及rAAV不同血清型和部分批间一致性。结果:CGE-LIF分析不同血清型rAAV(包括2、5、8、9、10型)可将其衣壳蛋白VP1、VP2和VP3基线分离并且峰形尖锐。准确性显示衣壳蛋白VP1、VP2和VP3的实测结果与预期结果的相关系数r>0.99;重复性结果显示每个衣壳蛋白VP1、VP2和VP3的迁移时间RSD均<1.5%,校正峰面积百分比的RSD均<5%;中间精密度结果显示衣壳蛋白VP1、VP2和VP3的迁移时间RSD均<1.5%,校正峰面积百分比RSD均<5%;线性结果显示在0.6×1012~3.0×1012 vg·mL-1范围内与衣壳蛋白校正峰面积线性关系良好;VP3检测限和定量限分别为7.2×1010 vg·mL-1和2.2×1011 vg·mL-1。用3种不同染料(P503、FQ和TAMRA)进行CGE-LIF检测,结果良好。该方法的批间稳定结果较为理想。结论:建立了rAAV衣壳蛋白CGE-LIF分析方法,所建方法可以对rAAV产品3种衣壳蛋白进行分离和相对定量。
Objective: To established a capillary gel electrophoresis tandem laser induced fluorescence detector (CGE-LIF) method to analyze the proportion content of recombinant adeno-associated virus (rAAV) capsid proteins VP1, VP2 and VP3, and to verify the methodology. Methods: The CGE-LIF analysis method for rAAV capsid protein was as follows: rAAV samples were pre denatured with a solution of 4% SDS-150 mmol·L-1 NEM (70 ℃, 5 min); labeled with P503 dye (70 ℃, 10 min); denatured with 1% SDS (70 ℃, 5 min), then the processed rAAV samples were mixed well and tested. The method validation included accuracy, repeatability, linearity, detection limit, quantification limit, durability, etc. In addition, the conditions of P503, TAMRA, and FQ dyes, as well as the consistency between different serotypes and between some batches of rAAV were investigated. Results: CGE LIF analysis of different serotype of rAAV (including types 2, 5, 8, 9, and 10) could separate their capsid proteins VP1, VP2, and VP3 at baseline and had sharp peaks. The accuracy showed that the correlation coefficient r was >0.99 between the measured results of capsid proteins VP1, VP2, and VP3 and the expected results. The repeatability results showed that the migration time RSD of each capsid protein VP1, VP2, and VP3 was less than 1.5%, and the peak area percentage RSD was less than 5%. The intermediate precision results showed that the migration time RSD of capsid proteins VP1, VP2, and VP3 were all less than 1.5%, and the peak area percentage RSD were all less than 5%. The linear result was displayed from 0.6×1012 vg·mL-1 to 3.0×1012 vg·mL-1, within the range, there was a good linear relationship with the peak area of capsid protein. The detection limit and quantification limit of VP3 were 7.2×1010 vg·mL-1 and 2.2×1011 vg·mL-1. Three different dyes (P503, FQ, and TAMRA) were used for CGE-LIF detection, and the results were good. The inter batch stability results of this method were relatively ideal. Conclusion: A CGE-LIF analysis method for rAAV capsid protein has been established, which can separate and quantify the proportion of three capsid proteins in rAAV products.
[1] GOSWAMI R, SUBRAMANIAN G, SILAYEYA L, et al. Gene therapy leaves a vicious cycle[J]. Front Oncol, 2019, 9(24):297
[2] WU Z, ASOKAN A, SAMULSKI RJ. Adeno-associated virus serotypes: vector toolkit for human gene therapy[J]. Mol Ther, 2006, 14(3): 316
[3] NASO MF, TOMKOWICZ B, PERRY WL, et al. Adeno-associated virus (AAV) as a vector for gene therapy[J]. Biodrugs, 2017, 31(4):317
[4] CARTER BJ. Adeno-associated virus and the development of adeno-associated virus vectors: a historical perspective[J]. Mol Ther, 2004,10(6):981
[5] EON-DUVAL A, BROLY H, GLEIXNER R. Quality attributes of recombinant therapeutic proteins: an assessment of impact on safety and efficacy as part of a quality by design development approach[J]. Biotechnol Prog, 2012, 28(3):608
[6] OYAMA H, ISHII K, MARUNO T, et al. Characterization of adeno-associated virus capsid proteins with two types of VP3-related components by capillary gel electrophoresis and mass spectrometry[J]. Hum Gene Ther, 2021, 32(21-22):1403
[7] WANG D, TAI P, GAO G. Adeno-associated virus vector as a platform for gene therapy delivery[J]. Nat Rev Drug Discov, 2019, 18(5):358
[8] PACOURET S, BOUZELHA M, SHELKE R, et al. A rapid and robust assay for batch-to-batch consistency evaluation of AAV preparations[J]. Mol Ther, 2017, 25(6): 1375
[9] WRIGHT JF. Manufacturing and characterizing AAV-based vectors for use in clinical studies[J]. Gene Ther, 2008, 15(11):840
[10] 基因治疗产品药学研究与评价技术指导原则 [S]. 2021
Technical Guidelines for Pharmaceutical Research and Evaluation of Gene Therapy Products[S]. 2021
[11] LI M, YU C, WANG W, et al. Interlaboratory method validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) methodology for analysis of mAbs[J]. Electrophoresis, 2021, 42(19):1900
[12] NUNNALLY B, PARK SS, HONG M, et al. A series of collaborations between various pharmaceutical companies and regulatory authorities concerning the analysis of biomolecules using capillary electrophoresis[J]. Chromatographia, 2006, 64(5):359
[13] TRICHT EV, GEURINK L, BACKUS H, et al. One single, fast and robust capillary electrophoresis method for the direct quantification of intact adenovirus particles in upstream and downstream processing samples[J]. Pure Appl Chem, 2017, 166(1): 8
[14] ZHANG CX, MEAGHER MM. Sample stacking provides three orders of magnitude sensitivity enhancement in SDS capillary gel electrophoresis of adeno-associated virus capsid proteins[J]. Anal Chem, 2017, 89(6):3285
[15] ZHANG Z, PARK J, BARRETT H, et al. Capillary electrophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection as a highly sensitive and quality control-friendly method for monitoring adeno-associated virus capsid protein purity[J]. Hum Gene Ther, 2021, 32(11-12):628
[16] BENNETT A, PATEL S, MIETZSCH M, et al. Thermal stability as a determinant of AAV serotype identity[J]. Mol Ther-Meth Clin D, 2017, 6(15): 171
