AAV process intensification by perfusion bioreaction and integrated clarification

João P Mendes; Bárbara Fernandes; Earl Pineda; Shashi Kudugunti; Mike Bransby; René Gantier; Cristina Peixoto; Paula M Alves; António Roldão; Ricardo J S Silva

doi:10.3389/fbioe.2022.1020174

AAV process intensification by perfusion bioreaction and integrated clarification

Front Bioeng Biotechnol. 2022 Nov 7:10:1020174. doi: 10.3389/fbioe.2022.1020174. eCollection 2022.

Authors

João P Mendes^{1

2}, Bárbara Fernandes^{1

2}, Earl Pineda³, Shashi Kudugunti³, Mike Bransby³, René Gantier³, Cristina Peixoto^{1

2}, Paula M Alves^{1

2}, António Roldão^{1

2}, Ricardo J S Silva^{1

2}

Affiliations

¹ iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
² Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
³ Repligen, Waltham, MA, United States.

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.

Keywords: adeno-associated virus; alternating tangential flow; gene therapy; integrated manufacturing; perfusion; process intensification; tangential flow depth filtration.