Abstract
Recent advances in gene therapy have increased the demand for large-scale manufacturing processes. The production of recombinant adeno-associated viral (rAAV) vectors, the predominant vectors used for gene delivery, requires efficient scalable processes for production and purification which have to comply with high safety standards. To enable operation in a fully closed system, the exclusive use of single-use materials and equipment is indispensable. The present study reports on the use of alluvial filtration for the clarification of lysed rAAV vector production culture from various production scales. In small-scale experiments (1-3 L feed), the in-house developed method was superior to the use of a synthetic depth filter regarding clarification performance for capacity, turbidity reduction, product recovery, and impurity reduction. Scale-up calculations for 500 L harvest revealed that alluvial filtration could provide the required filter area and capacity within a fully closed system, whereas the use of the depth filter could not. Real-life scale-up data for processing 10 L, 100 L, and 500 L with different rAAV vector variants confirmed scalability of the alluvial filtration method and yielded excellent results regarding its clarification performance. Moreover, the full filter capacity and limits of the system were not reached, indicating the robustness of the process to varying feedstock compositions. The safety of the established alluvial filtration train was supported by implementing enhanced monitoring and control to mitigate the risk of failure and leakages. Overall, the harvest of rAAV vectors by alluvial filtration proved to be superior to established depth filters within a biopharmaceutical production process. KEY POINTS: • Large-scale viral vector production (> 100 L) to meet increasing gene therapy demand • Alluvial filtration outperforms depth filters in efficient harvest clarification • Alluvial filtration offers capacity for closed-system scale-up to 500 L production.