AAV process intensification by perfusion bioreaction and integrated clarification

Abstract

Adeno-associated viruses (AAVs) demand for clinical trials and approved therapeutic applications is increasing due to this vector’s overall success and potential. The high doses associated with administration strategies challenges bioprocess engineers to develop more efficient technologies and innovative strategies capable of increasing volumetric productivity. In this study, alternating tangential flow (ATF) and Tangential Flow Depth filtration (TFDF) techniques were compared as to their potential for 1) implementing a high-cell-density perfusion process to produce AAV8 using mammalian HEK293 cells and transient transfection, and 2) integrating AAV harvest and clarification units into a single step. On the first topic, the results obtained demonstrate that AAV expression improves with a medium exchange strategy. This was evidenced firstly in the small-scale perfusion-mocking study and later verified in the 2 L bioreactor operated in perfusion mode. Fine-tuning the shear rate in ATF and TFDF proved instrumental in maintaining high cell viabilities and, most importantly, enhancing AAV-specific titers (7.6 × 104 VG/cell), i.e., up to 4-fold compared to non-optimized perfusion cultures and 2-fold compared with batch operation mode. Regarding the second objective, TFDF enabled the highest recovery yields during perfusion-based continuous harvest of extracellular virus and lysate clarification. This study demonstrates that ATF and TFDF techniques have the potential to support the production and continuous harvest of AAV, and enable an integrated clarification procedure, contributing to the simplification of operations and improving manufacturing efficiency.

High-cell-density production of adeno-associated viral vector serotype 6 by triple transfection in suspension HEK293 cell cultures

“…Recently, the improvement of AAV8 production at high-cell density with a medium exchange strategy was reported [64]. Conversely, the cell density effect was confirmed during our high-cell-density productions (Figures 3B and 5).…”

“…The cells were inoculated into the bioreactor in fresh medium at the desired cell density of 4 × 10 6 cells/mL to prevent detrimental effects from the spent medium. At larger scales, this could be achieved, for example, by medium exchange using Tangential Flow Depth Filtration (TFDF) as a cell retention device [63,64]. This bioreactor production resulted in 30-and 7.5-fold increase in VG/mL and VG/cell titers, respectively, compared to previously reported production at a similar scale [47].…”

Production of adeno‐associated viral vector serotype 6 by triple transfection of suspension HEK293 cells at higher cell densities

“…[69] Recently, the improvement of AAV8 production at high-cell density with a medium exchange strategy was reported. [65] Conversely, the CDE was confirmed during our HCD productions (Figures 3B and 5).…”

“…Recently, the improvement of AAV8 production at high‐cell density with a medium exchange strategy was reported. [ 65 ] Conversely, the CDE was confirmed during our HCD productions (Figures 3B and 5). CDE refers to the diminished production at high cell densities resulting from decreased cell‐specific productivity.…”

“…At larger scales, this could be achieved, for example, by medium exchange using a membrane‐based cell retention device, such as the alternating tangential flow (ATF) or the tangential flow depth filtration (TFDF) system. [ 64,65 ] This bioreactor production resulted in 30‐fold increase in VG mL −1 titer, compared to previously reported production at a similar scale. [ 47 ] Surprisingly, the viral vector yield on the bioreactor was around 10‐fold higher than the small‐scale satellite culture.…”