Impact of hydrodynamic, mechanical, and interfacial stress during downstream processing on adeno-associated virus...

Adeno-associated virus (AAV) is now well established as one of the most promising delivery vectors for gene therapy. During the manufacturing process, solutions of AAV are exposed to hydrodynamic and mechanical forces, and their impact on AAV integrity and product quality are still poorly understood. In this work, we studied the effects of different physical stress factors encountered during bioprocessing, including pumping, extensional flow, cavitation, mechanical stress, air-liquid interfaces, and high shear rates on two of the most employed serotypes, AAV8 and AAV9. The influence of stress factors was assessed in scale-down models, and the impact was determined by measuring soluble AAV concentration in solution, full-to-total capsid ratio, AAV aggregation and fragmentation, capsid molecular weight, capsid size, particle content, and thermal stability. Our data demonstrate that significant losses of soluble AAV occur through all operations that included pumping, and this effect was more pronounced for AAV8. This serotype also showed higher propensity for particle formation in response to the physical strains. None of the stresses resulted in the appearance of size or weight variants of soluble AAV, and no effect on capsid thermal stability or temperature of aggregation onset was observed. In addition, no changes in full-to-total capsid ratio were detected. Remarkably, both AAV8 and AAV9 experienced little impact and no reduction in titer when exposed to isolated high shear rates (20,000 s^-1). This illustrates the stability of AAV viral vectors towards shear forces but highlights the need to carefully optimize parameters for pumping operations to enhance performance and ensure consistency during AAV bioprocessing.