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 × 10⁶ 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 × 10¹³ virions were produced while a control experiment with an ATF system equaled to 1.9 × 10¹³ virions. For infection at 25 × 10⁶ 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 × 10⁶ 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 culture-based 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.

Keywords: continuous harvesting; inclined settler; influenza vaccine; perfusion; suspension cell culture.

FIGURE 1

Perfusion cell culture set-up using an inclined settler. Cells are recirculated in a loop using a peristaltic pump at a flow rate of 35 mL/min. At the top of the inclined settler, another peristaltic pump harvests cell-free medium. The addition of fresh medium through the feeding pump allows maintaining the working volume at steady state. Blue and red arrows indicate the flow direction (water recirculation) in the heat exchanger.

FIGURE 2

Growth and metabolism of AGE1.CR.pIX cells in perfusion mode using a stirred-tank bioreactor coupled to an inclined settler or an ATF system. Cultivations with an inclined settler (IS): IS1 (), IS2 (), IS3 (), IS4 (), IS5 (), and IS6 (). Cultivation with the ATF system (). (A) Viable cell concentration (filled symbols) and cell viability (empty symbols). (B) Doubling time (t_d) during the cell growth phase. (C) Cell-specific glucose consumption rate (q_glc) during perfusion (after 48 h). (D) Lactate yield based on glucose consumption (Y_lac/glc) during perfusion (after 48 h).

FIGURE 3

Production of influenza A virus in perfusion mode using AGE1.CR.pIX cells (time of infection t = 0 h). Cultivations in stirred-tank bioreactor with an inclined settler (IS) IS3 (), IS4 (), IS5 (), and IS6 () plus one control run with an ATF system () were carried out. (A, B) Cells were infected at 25 × 10⁶ cells/mL (IS3, IS4, and ATF) or (C, D) 50 × 10⁶ cells/mL (IS5, IS6). (A, C) Viable cell concentration (filled symbols) and cell viability (empty symbols) shown as average of analytical duplicates. (B, D) Perfusion rate in bioreactor working volume per day (day^–1).

FIGURE 4

Influenza A virus production in perfusion cultivations of AGE1.CR.pIX cells with inclined settler (IS) IS3 (), IS4 (), IS5 (), IS6 (), and ATF system (). (A, C) Concentration of virions in the bioreactor and in the harvest, based on HA titer; (B, D) concentration of infectious virions, based on TCID₅₀. The samples were taken from the bioreactor (filled symbols) and the harvest (empty symbols).

FIGURE 5

Progression of infection of cells with influenza A virus in perfusion cultivations determined by imaging flow cytometry. (A) Fraction of infected cells positive for virus nucleoprotein and (B) concentration of infected cells in the bioreactor, calculated from the measured total cell concentration and the fraction of infected cells. Runs: IS3 (), IS4 (), IS5 (), IS6 (), and ATF ().

FIGURE 6

(A) Cell growth and (B) total number of virions produced (Vir_tot, based on HA titer) after infection using an acoustic settler without heat exchanger (AS1, ) or with heat exchanger (AS2, ), compared to runs IS3 (), IS4 (), and ATF (). The total number of virions produced was normalized to a bioreactor working volume of 650 mL (see section “Yield and titer calculations”).