Investigating adeno-associated virus stabilization at the air-water interface through competitive adsorption.

This study investigated the stabilization of adeno-associated viruses (AAVs) at the air-water interface (AWI), focusing on the interplay between surfactant and capsid adsorption during agitation stress. AAV-9 and its engineered variant, AAV-PHP.eB-known for enhanced central nervous system tropism-were subjected to orbital shaking to simulate interfacial stress encountered during manufacturing and transportation. The impact of surfactant type and concentration was systematically evaluated using polysorbates 20 and 80, Poloxamer 188 (P 188), and Kolliphor® HS15. Size-exclusion chromatography-multi-angle light scattering (SEC-MALS) and total holographic characterization (THC) provided insights into capsid titer recovery and subvisible particle (SVP) formation. Hydrophobic interaction chromatography (HIC) and pendant drop shape analysis revealed striking differences in surface hydrophobicity and activity between the AAV variants. Our results suggested that AAV-PHP.eB, with higher surface hydrophobicity and activity, suffered greater adsorption and SVP formation than AAV-9, requiring higher P 188 concentrations for effective stabilization. At 0.1 % (w/v) P 188, a shift in SVP populations was detected by THC and Dynamic Light Scattering with the emergence of rod-like and irregularly shaped particles, likely representing P 188 aggregates. The findings suggest that greater P 188 concentration not only offers protection by saturating the AWI and direct binding to the capsid, but may also form aggregate structures in presence of AAVs that further impede capsid adsorption and aggregation.