Beaumal Corentin, Guapo Felipe, Smith Josh, Carillo Sara, Bones Jonathan
Characterisation and Comparability Laboratory, NIBRT - National Institute for Bioprocessing Research and Training, Foster Avenue, Belfield, Blackrock, Dublin, A94 X099, Ireland.
School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin, D04 V1 W8, Ireland.
Anal Bioanal Chem. 2025 Apr 21. doi: 10.1007/s00216-025-05874-4.
Adeno-associated virus (AAV) viral vector-based gene therapy is advancing rapidly, offering potential treatments for rare and severe diseases. The AAV capsid consists of a combination of three viral proteins (VPs), VP1, VP2, and VP3, ranging from 59 to 81 kDa and present at a theoretical bulk ratio of 1:1:10. This study employed hydrophilic interaction liquid chromatography (HILIC) and mass spectrometry (MS) to achieve robust separation and detailed characterisation of AAV9 capsid proteins. Advanced top-down MS approaches combining multiple fragmentation techniques (HCD, ETD, EThcD, and UVPD) were successfully applied, increasing the sequence coverage up to 40% for VP3 and confirming N-terminal acetylation on VP1 and VP3. The workflow demonstrated high reproducibility between injection duplicates and was subsequently applied to the characterisation of in-house produced biological replicates of AAV9 samples from HEK293 cells, showing consistent results across them. Analysis of AAV9 derived from Sf9 insect cells, a more complex sample due to higher levels of modification of the capsid VPs, further evidenced method versatility. Overall, this study highlights the potential of HILIC-MS and advanced top-down MS approaches for detailed characterisation of AAV capsid proteins.
基于腺相关病毒（AAV）载体的基因治疗正在迅速发展，为罕见和严重疾病提供了潜在的治疗方法。AAV衣壳由三种病毒蛋白（VP），即VP1、VP2和VP3组成，分子量在59至81 kDa之间，理论上的总体比例为1:1:10。本研究采用亲水相互作用液相色谱（HILIC）和质谱（MS）技术，实现了AAV9衣壳蛋白的有效分离和详细表征。成功应用了结合多种碎裂技术（HCD、ETD、EThcD和UVPD）的先进自上而下质谱方法，使VP3的序列覆盖率提高到40%，并确认了VP1和VP3的N端乙酰化。该工作流程在重复进样之间显示出高重现性，随后应用于来自HEK293细胞的AAV9样品的内部制备生物复制品的表征，各复制品结果一致。对来自Sf9昆虫细胞的AAV9的分析进一步证明了该方法的通用性，Sf9昆虫细胞来源的样品由于衣壳VP的修饰水平较高而更为复杂。总体而言，本研究突出了HILIC-MS和先进的自上而下质谱方法在详细表征AAV衣壳蛋白方面的潜力。
