Development of a scalable process for lentiviral vector mass production by transient transfection

Lentiviral vectors (LVs) are promising delivery vehicles for applications in gene therapy. Yet, the field still relies on inefficient and non-scalable production strategies. Current production methods will become a limitation in later clinical evaluation phases and for commercialization when large amounts of LVs need to be generated.;An optimized protocol for LV production was developed using a HEK293 cell line grown in suspension cultures. In this scalable process, production in perfusion mode addressed the low stability of functional LVs. Several strategies such as transfection at high cell density, selection of advanced medium formulations and addition of the expression-enhancing additive sodium butyrate resulted in significant improvements of volumetric and specific productivity. The overall yield in functional LVs was increased by 100-fold and similar results were obtained for small and bioreactor scale cultures, reaching maximum functional LV titers in the range of 108 transducing units (tu)/mL.;The production kinetics under improved conditions was then analyzed employing several LV quantification methods. After transfection, highest functional LV titers were reproducibly found 2 days post-transfection. The results also showed that LV quality, as the ratio of functional to total LV particles was generally low with only 1-4 % of the total viral particles (measured as the number of viral genomes) being functional, i.e. having the ability to transfer genetic information. This ratio was not constant over time when sodium butyrate was added after transfection. LV quality was also increased at higher harvest rates. Our results also indicate that the cytotoxic effects of VSV-G might be limiting for further yield improvements of the current LV production system.;LV production kinetics was also characterized using online monitoring of the permittivity signal in bioreactor productions. Permittivity measurements are valuable for identification of events such as budding of viral vectors as dielectric properties of the producing cells are affected during the viral release process. The evolution of the permittivity signal was analyzed and linked to the LV production kinetics to identify four key process transition phases after transfection which are characteristic of LV production.;In this work, we first reviewed current LV production methods and point out the constraints intrinsic to these protocols. To date, routine LV production is almost exclusively performed in small scale systems using adherent cells. These protocols will not be able to satisfy the anticipated future demands in LVs. For their widespread testing in clinical settings and for the production of LV-based therapeutics after their approval, novel scalable production strategies are needed to robustly produce these vectors at high yield.;The in depth process characterization of the LV system with offline LV quantification methods and online tools provided several avenues for process monitoring and further process optimization.;The developed LV production strategy is based on high cell density transfection. The approach delivers LVs at high yield in a timely manner and should be easily adaptable to other LV constructs.;The strategies developed in this work provide unprecedented yields of 1010-1011 tu/L, corresponding to 1012 -1013 viral genomes (vg)/L of production supernatant. Production kinetics and process evolution are well characterized, which will guide future yield optimization. As it facilitates the production of LVs, the developed process should thus enable the evaluation and use of these vectors as a therapeutic and should be an attractive option to generate LV for future clinical trials.