Anion-exchange chromatography separates structurally heterogeneous and low-potency particles in adeno-associated...

Recombinant adeno-associated virus (rAAV) manufacturing generates various particles in addition to full particles (FPs), posing a significant challenge for downstream purification. In this study, we applied anion-exchange chromatography (AEX) to resolve such particles of rAAV followed by the characterization of three fractions including an unidentified fraction, termed P3, that elutes at higher salt concentrations. Unexpectedly, density gradient ultracentrifugation (DGUC) clarified that P3 contained both empty particles (P3E) and full particles (P3F). No variation in genome packaging was observed between FPs and P3F, whereas zeta potential measurement showed that P3E and P3F exhibited increased negative surface charge compared with empty particles (EPs) and FPs in the earlier fractions (P1 and P2), respectively. Further characterizations using biolayer interferometry revealed the externalization of the negatively charged N-terminal region in viral protein 1 (VP1) of P3F in P3. In addition, P3F exhibited slightly elevated levels of deamidation, a critical quality attribute associated with vector potency, resulting in their reduced transduction efficiency. Notably, such P3F were not separated by DGUC solely. Collectively, AEX provides an effective strategy for isolating high-quality FPs by removing not only EPs but also structurally altered FPs with reduced potency.