Monitoring lentiviral vector production kinetics using online permittivity measurements

Abstract

Lentiviral vectors (LVs) are effective delivery vehicles that have been successfully used in gene and cell therapy. LVs are most commonly produced via the transient transfection of several plasmid constructs in adherent cell cultures. Recently, we described an efficient and scalable LV production in serum-free suspension cultures. To further facilitate the translation of LV-based interventions to the clinic, the robustness of the production process needs to be ensured to ultimately achieve a specified quality and quantity of LV production lots. However, routine processes are largely empirical, and strategies to monitor LV production kinetics in real-time have not yet been described.In this work, in situ real-time permittivity measurements were assessed to document the production of LVs. Characteristic process phases that were closely associated with LV production kinetics were identified. The permittivity signal evolution was interpreted by exploiting various independent online and offline monitoring measurements. Cellular membrane properties and, to a lesser extent, cell size were the main factors contributing to the permittivity variations. It is concluded that the permittivityrelated parameters can be used for the detection of viral release, allowing real-time assessment of process performance. The technology should thus greatly facilitate process development and optimization.Crown

Real‐time monitoring of influenza virus production kinetics in HEK293 cell cultures

“…However, all of the events occurring in a cell system during viral infection (virion entrance, viral protein synthesis, bud formation, and progeny viral release) could potentially affect the cell's dielectric properties and be detected at a macroscopic level. This was recently documented by our group with lentiviral productions using HEK293 suspension cell cultures 38…”

“…From the results established at small‐scale and considering the potential of the multifrequency permittivity scan to monitor viral productions,33, 37, 38 influenza kinetics have been extensively documented to detect infection‐cycle events using an on‐line signal. Both virion formation and cell death, including apoptosis and necrosis, are major cellular events that impact the membrane capacitance ( C M ) and intracellular conductivity (σ i ).…”

“…Dielectric spectroscopy has been increasingly used to monitor a wide range of bioprocesses, from mammalian cell culture to microbiological systems 28–36. Nevertheless, very few studies have described the application of this technology to viral production processes 33, 37, 38. However, all of the events occurring in a cell system during viral infection (virion entrance, viral protein synthesis, bud formation, and progeny viral release) could potentially affect the cell's dielectric properties and be detected at a macroscopic level.…”

Influence of HEK293 metabolism on the production of viral vectors and vaccine

“…Hence, perfusion is not only used for production at high cell density but also for continuous harvest of viral product. Ansorge et al reported sustained lentivirus perfusion production with viral titers between 10 7 and 10 8 IVP/ml using feeding rates of 1-2 vol/day [82]. Several authors showed the use of perfusion for retrovirus production (reviewed in [83]).…”

Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes

“…It has been implemented in batch and fed-batch cultures, as well as in packed bed reactors. This method has also been dedicated to monitor production levels of viruses (lentivirus, baculovirus) or proteins, but also evolution of cell size or of processes (Zeiser et al 2000;Ansorge et al 2010Ansorge et al , 2011Cannizzaro et al 2003). A single group applied this method to adherent CHO cells cultivated on macroporous microcarriers (Ducommun et al 2001(Ducommun et al , 2002.…”