The manufacture of AAV for gene therapy applications using a closed, semi-automated hollow-fiber bioreactor.

Abstract

Adeno-associated viral (AAV) vectors have been established as a safe and effective delivery vehicle for gene therapy. However, current methods for AAV production using adherent approaches are suboptimal due to their reliance on a substantial number of plastic-based flasks, manual labor, and a significant manufacturing footprint. Consequently, a protocol for generating AAV2 was developed on the Quantum, a semi-automated closed hollow-fiber bioreactor platform. In this system, Human Embryonic Kidney 293T cells were successfully expanded and transfected to produce an average crude AAV2 titer of 4.92 × 10¹⁴ viral particles and 6.81 × 10¹³ viral genomes from 1.2 L of harvested cell lysate. The application of a standard AAV downstream process confirmed normal processability of the material. A cost of goods model comparing the Quantum bioreactor with the current standard HYPERStack36 and Corning CellSTACK 10-layer systems demonstrated that the Quantum bioreactor reduced the number of open steps by more than 40-fold, production time by up to 3.6-fold (HYPERStack36) and 7.5-fold (CellSTACK 10-layer), and costs by up to 2-fold (HYPERStack36) and 20.7-fold (CellSTACK 10-layer). Therefore, the Quantum bioreactor is an effective alternative to plastic flasks for the manufacturing of AAVs at both R&D and early translational scale, as it reduces production time, operating costs, and process risk.