Jin Jing, Wang Wentao, Gao Tie, Dai Yangguang, Tao Lei, Ma Qiang, Wang Lijun, Li Xiu, Chen Hongxu, Zhou Yong
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 2, Tiantan Xili, Beijing 100050, China.
SCIEX, 5F, Building 1, 24 Yard, Jiuxianqiao Mid Road, Chaoyang District, Beijing 100015, China.
J Pharm Biomed Anal. 2025 Dec 15;266:117112. doi: 10.1016/j.jpba.2025.117112. Epub 2025 Aug 13.
Recombinant adeno-associated viruses (rAAVs) are pivotal gene therapy vectors due to their safety and stable transduction, yet comprehensive characterization of capsid post-translational modifications (PTMs)-critical for potency, immunogenicity, and manufacturing consistency-remains limited across production platforms. This study employs microflow LC-MS/MS coupled with electron-activated dissociation (EAD) to analyze PTMs in clinically relevant rAAV5 and rAAV9 serotypes produced via mammalian (HEK293) and insect (Sf9) cells, with parallel cellular-level evaluation of vector potency and infectivity, conducted under matched purity and capsid thermal stability conditions to isolate PTM-specific effects. Intact mass analysis revealed conserved N-terminal acetylation in VP1/VP3 across both platforms, while PTM profiling identified six distinct modification types, including deamidation, oxidation, and phosphorylation, with Sf9-derived vectors exhibiting 14 % more PTMs than HEK293-produced counterparts. Despite comparable purity and thermostability, HEK293-derived vectors demonstrated superior in vitro potency (1.9-fold higher eGFP expression) and lower physical-to-infectious particle ratios (P:I, 1.8-3.2-fold reduction), linking PTM patterns to enhanced infectivity. EAD fragmentation mapped isoaspartate (IsoAsp) formation to specific asparagine residues, implicating deamidation-driven instability, while analysis of four Sf9-produced rAAV9 batches revealed ≤ 5 % lot-to-lot variability in PTM site counts. Preliminary data identified low-variance PTM sites (e.g., N57, N452; coefficient of variation, CV ≤ 15 %) and IsoAsp levels (CV ≤ 10 %) as potential stability markers for batch consistency monitoring, though their definitive utility as critical quality attributes requires further validation. These findings establish serotype- and platform-specific PTM landscapes under controlled biophysical parameters, providing actionable insights for optimizing production systems and establishing PTM-driven quality control in gene therapy.
重组腺相关病毒（rAAVs）因其安全性和稳定转导性而成为关键的基因治疗载体，然而，衣壳翻译后修饰（PTMs）对于效力、免疫原性和生产一致性至关重要，在不同生产平台上对其全面表征仍然有限。本研究采用微流液相色谱-串联质谱（LC-MS/MS）结合电子激活解离（EAD）来分析通过哺乳动物（HEK293）和昆虫（Sf9）细胞生产的临床相关rAAV5和rAAV9血清型中的PTMs，并在匹配的纯度和衣壳热稳定性条件下对载体效力和感染性进行平行细胞水平评估，以分离PTM特异性效应。完整质量分析揭示了两个平台上VP1/VP3中保守的N端乙酰化，而PTM谱分析确定了六种不同的修饰类型，包括脱酰胺、氧化和磷酸化，Sf9衍生的载体比HEK293生产的对应物表现出多14%的PTMs。尽管纯度和热稳定性相当，但HEK293衍生的载体表现出更高的体外效力（eGFP表达高1.9倍）和更低的物理感染性颗粒比率（P:I，降低1.8-3.2倍），将PTM模式与增强的感染性联系起来。EAD片段化将异天冬氨酸（IsoAsp）的形成映射到特定的天冬酰胺残基，暗示脱酰胺驱动的不稳定性，而对四个Sf9生产的rAAV9批次的分析显示PTM位点计数的批次间变异性≤5%。初步数据确定低变异性PTM位点（如N57、N452；变异系数，CV≤15%）和IsoAsp水平（CV≤10%）作为批次一致性监测的潜在稳定性标志物，尽管它们作为关键质量属性的确切效用需要进一步验证。这些发现建立了在受控生物物理参数下血清型和平台特异性PTM图谱，为优化生产系统和在基因治疗中建立PTM驱动的质量控制提供了可操作的见解。
