Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode

Abstract

Influenza viruses have been successfully propagated using a variety of animal cell lines in batch, fed-batch, and perfusion culture. For suspension cells, most studies reported on membrane-based cell retention devices typically leading to an accumulation of viruses in the bioreactor in perfusion mode. Aiming at continuous virus harvesting for improved productivities, an inclined settler was evaluated for influenza A virus (IAV) production using the avian suspension cell line AGE1.CR.pIX. Inclined settlers present many advantages as they are scalable, robust, and comply with cGMP regulations, e.g., for recombinant protein manufacturing. Perfusion rates up to 3000 L/day have been reported. In our study, successful growth of AGE1.CR.pIX cells up to 50 × 10 6 cells/mL and a cell retention efficiency exceeding 96% were obtained with the settler cooled to room temperature. No virus retention was observed. A total of 5.4-6.5 × 10 13 virions were produced while a control experiment with an ATF system equaled to 1.9 × 10 13 virions. For infection at 25 × 10 6 cells/mL, cell-specific virus yields up to 3474 virions/cell were obtained, about 5-fold higher than for an ATF based cultivation performed as a control (723 virions/cell). Trypsin activity was shown to have a large impact on cell growth dynamics after infection following the cell retention device, especially at a cell concentration of 50 × 10 6 cells/mL. Further control experiments performed with an acoustic settler showed that virus production was improved with a heat exchanger of the inclined settler operated at 27 • C. In summary, cell culturebased production of viruses in perfusion mode with an inclined settler and continuous harvesting can drastically increase IAV yields and possibly the yield of other viruses. To our knowledge, this is the first report to show the potential of this device for viral vaccine production.

Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production

“…This is in contrast to other studies on influenza A virus, lentivirus, and adenovirus production, which demonstrated advantages regarding continuous virus harvesting in perfusion cultures. [ 23,28,29,34–36 ] Regarding the cell growth phase, a similar growth dynamics and a high cell viability (>90%) was achieved. Non‐toxic levels of released metabolites (<20 m m lactate and <3 m m ammonium) were observed regardless of the cell retention device, demonstrating a healthy state of the cells allowing high productivity after infection as already observed in previous studies [ 37–40 ] (Figure 2A–C).…”

“…(2020). [ 29 ] The IS was operated at a recirculation rate of 35 mL min −1 , intermittent vibration (15 s on, 10 min off) and with 30° angle. Water at a temperature of 27 °C was recirculated for heat exchange.…”

OP7, a novel influenza A virus defective interfering particle: production, purification, and animal experiments demonstrating antiviral potential

“…However, continuous virus harvest with membrane-based cell retention remains problematic due to potential blocking of the membrane (Genzel et al 2014 ). Alternatively, cell retention devices like an acoustic settler might be used for continuous virus harvesting (Coronel et al 2020 ; Granicher et al 2020 ).…”

Cell culture-based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

“…Further, it was described that the mentioned alternative cell retention systems do not allow for complete cell retention and some cells are lost over the course of cultivations. Additionally, their separation principles rely on cell settling and therefore longer recirculation times were observed (Chotteau 2015;Coronel et al 2020;Granicher et al 2020). In the present study, no cells could be detected in the permeate line of the VHU (data not shown) and the cells only spent a few seconds outside the STR during each pumping cycle of the ATF system.…”