Results and Discussion
Cell growth of Vero cells in suspension
cultures
Screening for commercial
media
Suspension adapted Vero cells grew well in shake flasks in IHM03 medium
up to a cell density of around 2 × 106 cells/mL and
with doubling times of around 48 h as previously reported (Shen et al.,
2019). IHM03 is an in-house medium developed and produced in small
batches by the NRC, supporting the growth and virus production of Vero
suspension cultures. As reported by Shen et al. and Rourou et al., media
composition is critical for successfully generating a suspension adapted
Vero cell line and no commercial media was able to support Vero cell
adaptation so far (Rourou et al., 2019; Shen et al., 2019). Despite
these recently reported adaptation successes with in-house media,
establishing a process using commercial media could reduce the risk of
lot-to-lot variations and would make the platform more amenable to work
under standard conditions when media supply is assured.
Therefore, efforts have been dedicated to assessing different commercial
media. After 20 passages of gradual adaptation in shake flasks, a Vero
cell line was obtained that was able to grow in MDXK medium (Xell AG)
supplemented with 4 mM Glutamax and which exhibited cell doubling times
of around 48-72 h (data not shown). Other commercial media that were
tested, but did not support growth of Vero cells in suspension culture
included VP-SFM, OptiPRO, FreeStyle 293 (Thermo Fisher Scientific, USA),
HyClone HyCell TransFx-H (GE Healthcare, USA), HEK GM (Xell AG, Germany)
and ProVeroTM -1 serum free medium (SFM) (Lonza, Switzerland).
Despite slightly slower growth rates in MDXK than in IHM03 in shake
flasks, the cells were able to grow to similar cell densities in batch
shake flasks with less formation of cell aggregates. Compared to other
mammalian suspension cell lines, like derivatives of HEK293 or CHO with
cell doubling times of 24 h, there is still great potential to develop
media that can support similar growth rates.
Cell growth in batch
bioreactor
The suspension adapted Vero cells showed similar growth in IHM03 medium
in 1L batch bioreactors as previously reported, reaching
1.78 × 106 cells/mL after 6 days (Shen et al., 2019).
Cell viability was above 99 % during the whole run. The doubling time
was around 51 hours for the entire batch process duration between cell
seeding and peak in maximum cell density, and around 40 hours for the
exponential cell growth phase between cell seeding up until 96 hours
(Figure 1 A). The substrates glucose and glutamine were almost
depleted at the end of the culture. Ammonia was produced throughout and
stayed below concentrations of 4 mM. Lactate production reached a
concentration of 24.5 mM after 96 hours, but declined thereafter which
can be explained by an uptake of lactate by the cells as previously
reported (Quesney et al., 2003).
Cell growths in MDXK medium was slightly slower and only reached
1.45 × 106 cells/mL after 7 days (Figure 1B). However, in contrast to the shake flask experiments, Vero cells in
MDXK medium showed a higher degree of aggregate formation in the
bioreactor compared to cells in IHM03. This could have led to an
underestimation of the cell count, which is also indicated by a higher
glucose consumption rate in MDXK. Cell viability again was above 99 %
throughout the process. As opposed to the previous run, glucose and
glutamine were depleted earlier and required feeding, adjusting glucose
to 2 g/L and glutamine to 2 mM once daily starting on day 2 for
glutamine and day 6 for glucose. Ammonia production was similar, never
exceeding concentrations of 4 mM. Lactate production, however, was
significantly higher and surpassed 50 mM at the end of the culture.
Lactate was not consumed by the cells in MDXK medium indicating
significant differences in the cell metabolism in the two media.
rVSV-ZEBOV production in shake
flask
rVSV-ZEBOV production experiments were initially carried out at a
smaller scale in shake flasks to test multiple conditions
simultaneously. In particular, the effects of varying multiplicities of
infection (MOI), which is the ratio of infectious particles to the
number of cells at the time of infection (TOI), as well as infections at
different cell densities and in different growth media were screened.
During the late stages of the cultivation when cell densities exceeded
1 × 106 cells/mL, cells started to adhere to the
surface of the shake flask and cell aggregates were formed, which made
it quite difficult to accurately determine the cell count. Therefore,
initial experiments to investigate the infection kinetics of rVSV-ZEBOV
in suspension adapted Vero cells, were carried out by seeding a single
cell culture in fresh medium at a cell density of
1 × 106 cells/mL and cells were infected immediately
thereafter.
Previous studies of rVSV-ZEBOV in adherent Vero cells and suspension
cultures of HEK293 cells have shown that infection at a reduced
temperature of 34 °C led to higher infectious titers compared to 37 °C
(Kiesslich et al., 2020)(Gélinas et al., 2019). Based on these studies,
the temperature was lowered to 34 °C after infection in all experiments
of this work involving rVSV-ZEBOV.
Multiplicity of
infection
Vero cells were infected with rVSV-ZEBOV at different MOIs and samples
were taken every 12 hours to determine infection kinetics
(Figure 2 ). For the selected range of MOI, peak production of
rVSV-ZEBOV occurred between 24 and 36 hours post infection (hpi) and the
titers were in the same range with
1.10 × 107 TCID50/mL,
1.05 × 107 TCID50/mL
and 1.58 × 107 TCID50/mL at an MOI of
0.001, 0.01 and 0.1, respectively. In all cases, the infectivity
declined after the maximum titer had been reached. Similar kinetics have
been observed for adherent growing Vero cells, however, the titers were
more than eight times higher in adherent cell experiments, for example
8.79 × 107 TCID50/mL at an MOI of 0.01
at 36 hpi (Kiesslich et al., 2020). Further, the cell density at the
time of infection was more than three times higher for the suspension
cultures than for the adherent cell cultures in the reported study,
indicating even lower virus production per cell. It is to mention that
adherent Vero cells were cultivated in commercially available media,
optimized for cell growth and virus production.
rVSV infections of adherent Vero cells typically lead to a very distinct
cytopathic effect, where cells become round shaped and eventually lift
off from the surface. Of note, since suspension cells are already round
shaped and not attached to a surface, the cytopathic effect induced by
rVSV infections was less noticeable in the early stages of infection and
only became more apparent when the cell diameter increased due to viral
replication and when cells started to die from lysis caused by viral
release.
Nevertheless, based on these experiments and in accordance with our
previous work, it was decided to continue all subsequent rVSV
experiments at an MOI of 0.01.
Cell density
rVSV-ZEBOV replication in Vero cells seeded at different cell densities
was investigated to evaluate the effect of varying cell densities and to
assess if the production yield was affected by the cell growth phase or
metabolite concentrations at the time of infection. Figure 3shows rVSV-ZEBOV infection at 1 × 106 cells/mL,
2 × 106 cells/mL and 4 × 106cells/mL. The viral infection kinetics were slower compared to the
previous experiments and infectious titers peaked at 48 hpi for all
cases, indicating considerable variation between experiments.
Additionally, maximum infectious titers were around three times higher
for the run at 1 × 106 cells/mL, reaching
3.28 × 107 TCID50/mL. Throughout the
time course of the experiment, titers were even higher at
2 × 106 cells/mL, but the maximum infectious titer was
not significantly elevated at 48 hpi. For the run with a seeding cell
density of 4 × 106 cells/mL, infectious titers of
rVSV-ZEBOV were significantly higher, reaching
1.32 × 108 TCID50/mL, exceeding
infectious titers obtained from adherent Vero studies in 6-well plates
(Kiesslich et al., 2020). In comparison, for rVSV-GFP produced in
suspension cell cultures of Vero cells at different cell densities in a
similar experiment, 3.8 times higher virus titers were obtained at
2.5 × 106 cells/mL compared to
0.87 × 106 cells/mL (Shen et al., 2019). But a further
increase in cell density from 2.5 × 106 cells/mL to
5 × 106 cells/mL did not result in higher infectious
titers.
These results indicate that suspension cultures of Vero cells could be a
viable alternative if high cell density processes of suspension cultures
can be achieved at larger scale, but further research is necessary to
investigate effects of high cell density in more detail. One advantage
though is that suspension cultures are not limited by the surface area,
as is the case for adherent cell cultures using microcarriers, roller
bottles or fixed-bed bioreactors, which is the prevalent mode of virus
production in Vero cells. Nevertheless, these results need to be
carefully evaluated since it might be challenging to seed bioreactors in
fresh medium at high cell density
rVSV-ZEBOV, rVSV-HIV, and
rVSVInd-msp -SF-Gtc shake
flask production in different
media
Next, the kinetics of three variants, namely rVSV-ZEBOV, rVSV-HIV, and
rVSVInd-msp -SF-Gtcwere compared and the effect of different media on rVSV production were
studied. Based on temperature study results of rVSV-ZEBOV infections in
Vero cells, rVSV-HIV infections were carried out at 34 °C in a recent
study and this condition was adopted for this work as well (Mangion et
al., 2020). The
rVSVInd-msp -SF-Gtcconstruct, however, is temperature sensitive and therefore all
infections were carried out at 31 °C.
The IHM03 and MDXK adapted cell lines were infected with rVSV to compare
the virus production capacity of both media. Despite higher titers
obtained at higher cell densities (Figure 3 ), these experiments
were carried out at a seeding cell density of 1 × 106cells/mL to avoid cell aggregation. Figure 4 shows rVSV-ZEBOV
(A), rVSV-HIV (B) and
rVSVInd-msp -SF-Gtc (C)
replication in the MDXK adapted cell line at an MOI of 0.01 in
comparison to the corresponding experiment conducted in IHM03.
rVSV-ZEBOV replication in MDXK medium reached slightly higher titers
than in IHM03 medium (Figure 4 A). Where the infectious titer
reached a maximum at 24 hpi in IHM03, the titer in MDXK was with
2.63 × 107 TCID50/mL around 2.5 times
higher. Otherwise, almost identical infection kinetics were observed.
rVSV-HIV replicated better in IHM03 with a higher maximum titer of
9.59 × 106 TCID50/mL reached in a
shorter period of time (24 hpi) compared to
2.08 × 106 TCID50/mL in MDXK after 36
hpi (Figure 4 B). Titers of rVSV-HIV were lower in both media
than rVSV-ZEBOV and the infectivity declined faster than for rVSV-ZEBOV.
Besides, rVSV-HIV produced in adherent Vero cells reached up to
3.91 × 107 TCID50/mL at MOI of 0.01.
However, the peak was reached significantly later at 96 hpi (Mangion et
al., 2020).
In contrast to these two strains,
rVSVInd-msp -SF-Gtc reached
significantly higher titers (Figure 4 C). In MDXK,
5.19 × 108 TCID50/mL were reached at
36 hpi. In IHM03, almost two-fold higher titers with
1.17 × 109 TCID50/mL were reached.
However, it took additional 24 hours to reach this titer, which aligns
with the beginning of the replication phase being delayed. In addition
to higher infectious titers compared to rVSV-ZEBOV and rVSV-HIV, the
infectivity of viral particles did not decline significantly over the
following sample time points.
Higher infectious titers of
rVSVInd-msp -SF-Gtc in Vero
cells could be linked to the use of different transmembrane domains.
Whereas rVSV-ZEBOV and rVSV-HIV use Ebola GP or SIV transmembrane
domains (Mangion et al., 2020),
rVSVInd-msp -SF-Gtc is
expressing the native VSV-G protein transmembrane domain gene. It has
been shown that the stem region of the VSV-G glycoprotein was important
for efficient virus assembly, and viruses with shortened sequences were
replicated up 20-fold less (Robison and Whitt, 2000). More research
investigating the use of different transmembrane domains with the same
extracellular domain could reveal interesting aspects on virus
replication rates and identify new targets to improve the rVSV platform.
Nevertheless, it might be more appropriate to compare replication of
rVSVInd-msp -SF-Gtc to
rVSV-GFP production, which also uses the native VSV-G protein
transmembrane domain, and where titers of up to
8.93 × 109 TCID50/mL have been
obtained in shake flask experiments.
Besides, the lower process temperature of 31 °C is likely affecting the
infectivity. For example, the infectivity of this strain was not
declining over the following 36 h after the peak titer had been reached
thus potentially stabilizing infectivity. Though for rVSV-ZEBOV, an
optimal production temperature of 34 °C was determined in adherent Vero
cells (Kiesslich et al., 2020),
rVSVInd-msp -SF-Gtc is
based on VSVInd(GML) which was adapted to replicate well
at the lower temperature of 31 °C (Figure 4 C).
Bioreactor processes of suspension adapted Vero
cells
Bioreactor production of
rVSV-ZEBOV
Two bioreactors of Vero cells were infected at a cell density of
1.37 × 106 cells/mL and
1.02 × 106 cells/mL after cells grew for 4 days in
IHM03 and MDXK medium, respectively (Figure 5 ). The cell growth
phase corresponded well to the data shown in Figure 1 . Despite
a lower cell density at the TOI, maximum infectious titers were with
3.87 × 107 TCID50/mL more than one log
higher than in IHM03, were only 3.55 × 106TCID50/mL were obtained. In addition, replication was
faster in MDXK, were peak production occurred at 24 hpi compared to 36
hpi, respectively. The MDXK bioreactor also exhibited an almost 15-times
higher cell specific productivity with 37.9 TCID50/cell
compared to 2.6 TCID50/cell. Moreover, the ratio of
total viral particles to infectious particles was lower in MDXK (282
VG/TCID50) than in IHM03 (817
VG/TCID50), further indicating a better quality of the
final product if harvested at the time of peak infectious titer.
Compared to the shake flask experiments, rVSV-ZEBOV replication in MDXK
medium reached a slightly higher maximum infectious titer, indicating a
successful scale-up. For IHM03, titers were three times lower than in
shake flask and the peak was reached 12 hours later.
Further, in comparison to adherent Vero bioreactor productions of
rVSV-ZEBOV, the production using suspension adapted Vero cells in MDXK
appears superior. The infectious titer and the cell specific
productivities were slightly higher compared to the production in a
microcarrier bioreactor (1.42 × 107TCID50/mL, 7.6 TCID50/cell) and a
fixed-bed bioreactor (2.59 × 107TCID50/mL, 11.2 TCID50/cell). The ratio
of total viral particles to infectious particles was four times lower
than in the microcarrier but nine times higher compared to the fixed-bed
process (Kiesslich et al., 2020). Overall, the suspension Vero system is
a viable alternative to the current Vero manufacturing system carried
out in roller bottles for this Ebola virus disease vaccine (Monath et
al., 2019).
When set side by side to a suspension bioreactor production of
rVSV-ZEBOV in HEK293-SF, where a maximum of 1.19 × 108TCID50/mL was reached, production in Vero cells in MDXK
was 3 times lower. However, it can be expected that future media
development, bioprocess and cell line engineering of suspension Vero can
lead to significantly higher titers comparable to HEK293-SF (Gélinas et
al., 2019).
Bioreactor production of
rVSV-HIV
Two bioreactors were prepared as before, and Vero cell growth phase in
IHM03 and MDXK medium was consistent with the data from Figure
1 and Figure 5 . The two cultures were infected with rVSV-HIV
after 4 days (Figure 6 ). In contrast to the previous
experiment, virus production was favoured in IHM03 over MDXK. In IHM03,
rVSV-HIV reached a maximum titer of 2.12 × 107TCID50/mL, which was 25-times higher than in MDXK.
However, production kinetics of viral genomes were almost identical.
This is supported by a lower ratio of 143 VG/TCID50(IHM03) compared to 7041 VG/TCID50 (MDXK). Additionally,
the fact that the cell specific productivity in MDXK was 0.9
TCID50/cell implies that the rVSV-HIV/MDXK system failed
to scale-up and is not an adequate production system.
Besides, the shake flask experiment (Figure 4 B) already
indicated the superiority of IHM03 for rVSV-HIV replication. Moreover,
bioreactor production of rVSV-HIV in IHM03 exceeded titers from the
smaller scale, whereas bioreactor titers in MDXK subsided the small
scale.
Bioreactor production of
rVSVInd-msp -SF-Gtc
Finally,
rVSVInd-msp -SF-Gtcproduction in bioreactors of Vero suspension cell cultures was studied
(Figure 7 ). Again, the cell growth phase was comparable to the
previous runs, and cells were infected after 4 days. Infectious titers
of rVSVInd-msp -SF-Gtcpeaked in both media at 48 hpi. The beginning of the replication phase
was delayed in IHM03, as already seen in the shake flask experiments.
Nevertheless, titers were similar with 2.38 × 109TCID50/mL in IHM03 and 3.59 × 109TCID50/mL in MDXK. Thus, results from the shake flask
experiment were exceeded by two-fold and seven-fold, respectively. On
the one hand, the quality in terms of total viral particles to
infectious particles was with 3.0 VG/TCID50 better in
IHM03 than in MDXK, where this value was 6.0 VG/TCID50.
On the other hand, the cell specific productivity was doubled in MDXK
compared to IHM03, with 3670 TCID50/cell and 1803
TCID50/cell, respectively.
In comparison, rVSV-ZEBOV and rVSV-HIV productions in the bioreactor
peaked earlier. However, the number of viral genomes continued to
increase in those experiments even when the infectious titer declined.
Therefore, the rate of viral degradation is higher than the viral
production rate after the corresponding peak was reached in the case of
these two strains, potentially due to the higher process temperature of
34 °C versus 31 °C and its impact on viral stability.
Overall, the scale-up of
rVSVInd-msp -SF-Gtcproduction to the bioreactor was successful, exceeding small scale
results. In addition, this strain appears to replicate to much higher
titers, with a superior cell specific productivity and an improved ratio
of VG/TCID50 as compared to rVSV-ZEBOV and rVSV-HIV.