High-recovery AAV clarification using a multiplexed spiral inertial microfluidic platform

Abstract

The clinical adoption of emerging cell and gene therapies relies on the efficient upstream bioprocessing and high-recovery harvesting of viral vectors. AAV purification from cell culture begins with the removal of cells, through a process called cell clarification, which is typically performed with a membrane filter. Cells and debris accumulate on the filter matrix during clarification, resulting in filter clogging and significant product loss. Here, we implement spiral inertial microfluidic technology as an alternative, membraneless clarification strategy to separate AAVs from the host cells. We assemble a parallelized plastic spiral microfluidic system that divides an input flow rate of cell culture across 100 spirals to harvest clarified material at 20 mL/min. The microfluidic clarification platform achieves a viral vector recovery of 85%, with the additional 15% of recoverable AAVs remaining in the system. However, up to 20% of cells were also found in the harvest, signifying that cell removal by inertial clarification is incomplete. We incorporate spiral microfluidics with the 3M Harvest RC filter (0.2 µm) (3M, USA) into a two-step harvest process, where spiral microfluidic clarification is used first to remove most of the cells from the culture. Performing spiral microfluidic clarification before depth filtration improves the filter throughput by 56.25%, from 44.8 L/m² to 70 L/m² and leads to a two-step AAV recovery of ~74%, exceeding the 40 to 70% clarification recoveries generally reported. Our results support that further optimization and scale-out of the spiral inertial microfluidic system could increase the overall clarification recovery of AAV production processes.