Kontogiannis Theodoros, McElroy Christopher, Quaglia Milena, Foy Carole, Whale Alexandra S, Braybrook Julian, Smales C Mark
School of Natural Sciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
National Measurement Laboratory at the Laboratory of the Government Chemist (LGC), Guildford, Surrey GU2 7XY, UK.
Mol Ther Methods Clin Dev. 2025 Aug 13;33(3):101562. doi: 10.1016/j.omtm.2025.101562. eCollection 2025 Sep 11.
Accurate quantification and characterization of recombinant adeno-associated virus (rAAV) capsid proteins are critical for evaluating product quality and safety, ensuring batch consistency, and informing process development of their manufacture. The capsid consists of three proteins derived from the same gene, and while the mean capsid stoichiometry is nominally 1:1:10 (VP1:VP2:VP3), capsids with different stoichiometries exist. Recent studies show that variations in the capsid stoichiometry can impact vector infectivity. Here, a mass spectrometry (MS)-based method was developed to quantify VP1, VP2, and VP3 in rAAV9 capsids and determine stoichiometry. Additionally, the methodology delivers precise measurement of total capsid content and provides a greater depth of information than traditional ELISA capsid titer measurements. The method could be further refined as a reference method to standardize measurements and assign values to reference materials. Host cell proteins consistent with other findings reported in the literature were also identified and reported. The consistent detection of these host cell proteins across different studies highlights their potential relevance to gene therapy products and the importance of their monitoring. Our report exhibits the utility of MS for precise rAAV characterization and presents the first approach to using MS for the standardized measurement of rAAV across different drug products.
准确量化和表征重组腺相关病毒（rAAV）衣壳蛋白对于评估产品质量和安全性、确保批次一致性以及为其生产的工艺开发提供信息至关重要。衣壳由源自同一基因的三种蛋白质组成，虽然衣壳的平均化学计量比名义上为1:1:10（VP1:VP2:VP3），但存在不同化学计量比的衣壳。最近的研究表明，衣壳化学计量比的变化会影响载体的感染性。在此，开发了一种基于质谱（MS）的方法来量化rAAV9衣壳中的VP1、VP2和VP3并确定化学计量比。此外，该方法能够精确测量衣壳的总含量，并且比传统的ELISA衣壳滴度测量提供更深入的信息。该方法可进一步完善为一种参考方法，以标准化测量并为参考材料赋值。还鉴定并报告了与文献中其他研究结果一致的宿主细胞蛋白。在不同研究中对这些宿主细胞蛋白的一致检测突出了它们与基因治疗产品的潜在相关性以及监测它们的重要性。我们的报告展示了质谱用于精确表征rAAV的实用性，并提出了第一种使用质谱对不同药品中的rAAV进行标准化测量的方法。
