3.2. Evaluation of sorted cell pools under batch and pseudoperfusion modes
In order to compare cell growth and VLP production of cell pools 1 and 2, cells were cultivated in shaken spin tubes under batch (Figure 2A) and pseudoperfusion modes (Figure 2B). Spin tube cultures carried out under pseudoperfusion mode have been shown to serve as a good scale-down model for perfusion processes, reaching high viable cell concentrations comparable to those achieved in a continuous perfusion bioreactor process, with the added advantages of much lower media expenditure and possibility of simultaneously screening many different conditions before moving to bioreactor scale (Nikolay et al., 2020a).
Regarding cell growth in batch mode, cell pools 1 and 2 reached maximum viable cell concentrations of 7.6 and 9.9 million cells/mL, respectively (Figure 2A). Cells of pool 2 grew quicker, which led to earlier glucose depletion (Figure 2C) and a consequent earlier drop in cell viability when compared to cell pool 1. When both cell pools were grown in pseudoperfusion mode (PP) with daily medium exchange from day 4 on, much higher cell concentrations were obtained for both cell pools reaching maxima of approximately 40 and 50 million viable cells per mL for cell pools 1 and 2, respectively (Figure 2B). Enhanced cell growth in pseudoperfusion was enabled not only by the daily feeding of fresh medium (Figure 2D), but also by daily harvesting of the spent medium, promoting removal of any inhibitory metabolites. Since a fresh stock of nutrients was provided daily, the duration of the pseudoperfusion cultures was longer and could have been even longer if more than one medium exchange per day had been performed, but in these exploratory experiments we limited the medium exchange rate to 1 vvd (1 complete medium exchange per day).
Concerning product formation, maximum VLP concentrations in batch cultures were 98 and 163 mg/L for cell pools 1 and 2, respectively (Figure 2E). In pseudoperfusion, in spite of daily product harvesting, the high cell densities achieved allowed that high amounts of product were produced daily, so that maxima of 118 and 270 mg/L were achieved in PP for cell pools 1 and 2, respectively. Based on the volumes harvested at the end (10 mL) of batch cultures, or on day 4 (12 mL) and on all the following days (9 mL/day) for PP, cell pool 1 in batch mode resulted in a total harvest of 1.0 mg of VLPs on day 11, whereas in PP 9.0 mg were harvested altogether from day 4 to day 17. In the case of cell pool 2, batch culture resulted in 2.2 mg of VLPs, and PP culture allowed an accumulated harvest of 16.2 mg of VLPs. These data show that by doing a second FACS round it was possible to approximately double VLP production, both in batch and in PP. On the other hand, by moving from batch to PP allowed an average increase of 8.3 fold in total VLP produced for both cell pools. By combining both strategies – FACS enrichment of cell pools and pseudoperfusion operation – an increase of approximately 16.5-fold in total VLP production was achieved, for a process just somewhat longer (11 days for cell pool 1 in batch, as compared to 14 days for cell pool 2 in PP). Thus, the present data demonstrate that both strategies exploited herein contribute to process intensification and, if combined, can significantly increase VLP production and decrease production costs.