MS-based workflow for PTM profiling of rAAV capsid proteins to support bioprocess monitoring and development

Post-translational modifications (PTMs) of recombinant adeno-associated virus (rAAV) capsids have become a focus of interest due to their potential role in vector structure, stability, and function. However, consistent and comprehensive PTM profiling of rAAV capsid proteins remains challenging, especially for non-purified process intermediates. Here, we report the development and application of a robust workflow combining optimized sample preparation, mass spectrometry-based peptide mapping and PTM profiling tailored for rAAV analysis. Using a purified rAAV8 sample as a reference standard, we achieved >80% of MS/MS viral protein (VP) sequence coverage and quantified multiple PTMs, including N-terminal acetylation, deamidation, oxidation, and phosphorylation. This method showed high reproducibility and repeatability, with intra-assay and inter-assay variability within the expected range for label-free site-specific PTM relative quantification (5.5% and 25.1%, respectively). Importantly, we demonstrated the successful application of this workflow to in-process rAAV8 samples collected at different purification stages, revealing dynamic changes in PTM prevalence that might reflect process-related effects on capsid properties. Extending the method to rAAV5 samples demonstrated its robustness across serotypes. Stress studies further underscored the method's sensitivity to detect PTM shifts, while a stress-prone digestion protocol highlighted the importance of optimized conditions to minimize artificial modifications. Collectively, this work demonstrates a versatile analytical platform capable of reliable PTM monitoring in both purified and in-process rAAV samples, supporting its application in product characterization, process monitoring, and development for gene therapy manufacturing. • MS based MAM provides insights into site-specific PTMs of rAAV capsid proteins • Applicable to in-process samples, allowing monitoring across downstream stages • High reproducibility and repeatability demonstrated for rAAV8 analysis • Extended to rAAV5, shows robustness across different AAV serotypes • Provides insights into process impurities through host cell protein profiling