Next-Gen Protein Sequencing™ Enables Accurate AAV Serotype Profiling and Mixture Resolution for Gene Therapy 

Adeno-associated viruses (AAVs) are widely used as delivery vectors in clinical gene therapy, with each serotype exhibiting unique capsid protein sequences that influence tissue tropism, transduction efficiency, and immune response. Ensuring the identity and purity of AAV preparations is essential during manufacturing and quality control, particularly when closely related serotypes are involved. In this study, we demonstrate the use of Next-Gen Protein Sequencing™ (NGPS™) to identify AAV8 and AAV9 capsid proteins by detecting three serotype-specific peptides capturing amino acid differences at position 105, 315, 539, and 540. NGPS enabled reliable identification of each serotype and unambiguous detection of each serotype from a mixture, with sensitivity sufficient to detect AAV8 and AAV9 at levels of 10% in a mixture based on distinct kinetic signatures. These results highlight the utility of NGPS as a high-resolution analytical tool for AAV serotype characterization and monitoring of contamination.