Microflow LC-MS/MS reveals platform-specific post-translational modification signatures in recombinant adeno-associated virus capsids linked to enhanced potency and stability: A quality control framework for gene therapy.

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.