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.