3.3. Perfusion runs using XCellTM ATF-2
and inclined cell settler as cell retention devices
Based on the promising results of pseudoperfusion cultures obtained at
spin tube scale, perfusion bioreactor cultivations were carried out. At
first, a XCellTM ATF-2 was chosen as cell retention
device, since it has been widely used not only for production of
recombinant biopharmaceutical proteins, such as monoclonal antibodies
(Clincke et al., 2013), but has also been reported in the recent years
for virus (Coronel et al., 2019; Nikolay et al., 2018) and VLP
production processes (Lavado-García et al., 2020; Alvim et al., 2019).
In our previous work on zika VLP production (Alvim et al., 2019),
although a perfusion run could be stably operated for 30 days at
approximately 30 million cells/mL, product retention inside the
bioreactor due to membrane fouling was observed. However, considering a
recent report (Nikolay et al., 2020b) that showed a polysulfone hollow
fiber cartridge to be highly permeable for the yellow fever virus, we
decided to use a cartridge of hollow fibers having the same
characteristics as reported by these authors, with the aim of avoiding
fouling.
Cell pool 1 was inoculated at 0.5 × 106 cells/mL and
maximum viable cell density (VCD) was 28.8 × 106cells/mL on day 13, as measured offline by trypan blue exclusion in a
cell counter. We also monitored VCD online using a capacitance-based
biomass sensor. As shown in Figure 3A, the profiles of viable cell
density determined using both methods were very similar, with the online
biomass sensor showing a trend to be somewhat more sensitive. The use of
this sensor can be very important in processes where the cell-specific
perfusion rate is controlled, since the online biomass data can be used
to automatically adjust the fresh medium feed rate (Dowd et al., 2003).
Cell viability remained above 90% until day 16 (Figure 3A), but then
decreased because of nutrient depletion (Figure 3B). Until day 10,
perfusion rate had been gradually increased along time in order to meet
nutrient demands by the growing cell concentration. However, by the time
viable cell density was approximately 25 million cells/mL, no increases
in permeate withdrawal rate beyond 0.45 vvd were possible anymore
(D_harvest, Figure 3B) due to progressive fouling of the membrane
cartridge. Because the rate of permeate withdrawal was limiting, fresh
culture medium addition rate (D_feed, Figure 3B) for 3 consecutive days
exceeded the permeate and affected bioreactor working volume. Even so,
from day 15 on glucose exhaustion in the bioreactor occurred, causing
cell death. Another evidence that membrane fouling was progressively
occurring was the fact that product retention inside the bioreactor
started being observed from day 12 on, causing an increase in VLP
concentration inside the bioreactor and a decrease in VLP recovery in
the harvest (Figure 3C).
As discussed above, product retention had already been observed for
virus and VLP production when typical polyethersulfone ATF cartridges
with 0.2 μm pore size and 0.13 m2 filtration area were
used (Alvim et al., 2019; Coronel et al., 2019; Lavado-García et al.,
2020). In spite of using in the present work a different membrane
cartridge, having the same characteristics (polysulfone membranes with
0.4 µm pore size and 0.005 m² filtration area) as the cartridge that
Nikolay et al. (2020b) found to promote an efficient yellow fever virus
recovery, product retention did occur again.
In their work, Nikolay et al. (2020b) tested the cartridge in a
tangential-flow filtration experiment lasting just a few hours, whereby
a bioreactor with YFV-infected BHK-21 cells set in a closed
recirculation loop was just used to provide cell/virus suspension to
challenge the membrane cartridge – no perfusion cultivation was carried
out. Thus, considering the results obtained in the present work, it
seems probable that in order to effectively avoid fouling in a long-term
perfusion process, a membrane cartridge with the characteristics
selected by Nikolay et al. (2020b) would need to have a much larger
filtration area, such as for example the 0.13 m2 of
the traditional cartridge sold by the ATF manufacturer for the ATF-2
system.
Thus, to overcome product retention and membrane fouling issues observed
in ATF-based perfusion herein, large-area membrane cartridges of
specific materials and porosities could possibly be a solution and
should be tested. However, in view of the low production cost that is
desirable for a vaccine to be accessible also for the population of
low-income countries, we considered it more adequate to proceed
investigations using an inclined lamella settler, which is a low-cost
cell retention device that does not use membranes or any other
consumables and is intellectual-property free. Although no previous
reports of HEK293 perfusion processes using a settler was found in
literature, this cell retention device has been shown to provide very
high separation efficiencies with no product retention in previous works
with other mammalian cell lines (Coronel et al., 2020a; Coronel et al.,
2020b).
As shown in Figure 4, a perfusion run with a CS-10 inclined settler
using cell pool 2 resulted in a maximum VCD of
41.2 × 106 cells/mL on day 12, thus reaching the same
viable cell concentration as obtained in pseudoperfusion on the same
cultivation day (Figure 2B). Medium exchange rate was gradually
increased during the run, with the aim of maintaining glucose levels at
approximately 1 g/L (Figure 4B). In order to sustain VCDs above 25
million cells/mL, perfusion rates above 1.5 vvd had to be set,
confirming how importantly membrane fouling in the ATF-based perfusion
affected the necessary nutrient supply. Regarding the VLP product, as
expected no product retention was observed, and VLP concentrations
inside the bioreactor and in the perfusate harvest were of approximately
200 mg/L from day 10 on (Figure 4C) at dilution rates of up to 2 vvd
(Figure 4B), thus reaching volumetric productivities of approximately
400 mg/L/d, which are higher than those obtained in the pseudoperfusion
run (270 mg/L at 1 vvd, thus volumetric productivity of 270 mg/L/d).
Based on these results, the use of the inclined cell settler combined to
the double-sorted cell pool 2 holds a promising potential to establish
an intensified perfusion process for yellow fever VLP production.