Vaccine 38 (2020) 3639–3645

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Development of a high-yield live-virus vaccine production platform

using a novel fixed-bed bioreactor
Dalton M. Berrie a, Robin C. Waters a, Christopher Montoya a, Alex Chatel b, Eric M. Vela a,⇑
a
Ology Bioservices, Process Development, 13200 NW Nano Ct., Alachua, FL 32615, USA
b
Univercells, Rue de la Maîtise 11, 1400 Nivelles, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: The increasing importance of viral vaccine manufacturing has driven the need for high cell density pro-
Received 16 December 2019 cess optimization that allows for higher production levels. Vero cells are one of the more popular adher-
Received in revised form 17 March 2020 ent cell lines used for viral vaccine production. However, production is limited due to the logistical
Accepted 22 March 2020
limitations surrounding adherent cell line processes, such as large equipment footprints, time and
Available online 1 April 2020
labor-intensive processes, and larger costs per dose. We have addressed this limitation with the estab-
lishment of a viral vaccine production system utilizing the novel single use scale-XTM carbo bioreactor.
Keywords:
The unit is compact and is scalable and one of the novel features of the system is the continuous in-
Vesicular stomatitis virus
Fixed-bed bioreactor
line downstream purification and concentration processes associated with the bioreactor vessel. We pre-
Vaccine development sent the results from a campaign featuring a proprietary Vero cell line for production of a live recombi-
scale-X carbo nant Vesicular stomatitis virus vaccine that features the Lassa Fever virus glycoproteins. Metabolite
analyses and viral yield comparison between traditional flasks, cell factories, and the scale-X carbo biore-
actor were performed, and on average, the single use bioreactor produced 2–4 logs higher titers per sur-
face area, approximately 5
1010 pfu/cm2, compared to classical flatstock, 2.67
106 pfu/cm2, and cell
factories production, 5.77
108 pfu/cm2. Overall, we describe a novel bioreactor platform that allows for
a cost-efficient and scalable process for viral vaccine production.
Ó 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Single-use, fixed-bed bioreactors have emerged as platforms for
employing adherent cells for production of a myriad of various
viral vectors, live viruses, and virus-based vaccines. These bioreac-
tors combine a low shear environment of flatware systems with
automation and scalability. The Pall iCELLisÒ and Eppendorf
Fibra-CelÒ systems have been used to produce high yields of viral
vectors and live viruses [1–4]. There are significant advantages
for utilizing these closed fixed-bed bioreactor systems, which
include product containment, limited resources and materials,
time savings for production, process flexibility, and lower per-
dose production costs [5–7]. A recent fixed-bed entrant into the
market is the scale-XTM bioreactor system from Univercells. The
scale-X portfolio offers a range of growth surfaces: scale-X ‘hydro’
(<3 m2), ‘carbo’ (10–30 m2), and ‘nitro’ (200–600 m2). This range
offers a scalable process and the capability for clinical lot produc-
tion. Within the Univercells product line, the bioreactor height
⇑ Corresponding author.
E-mail address: eric.vela@ologybio.com (E.M. Vela).
increases, while the diameter is held constant. For example, the
carbo 10 m2 bioreactor is 1/3 the height of a 30 m2 bioreactor.
However, scale-up among the different lines is achieved by keeping
the height of the fixed-bed constant and increasing the diameter,
similar to scale-up in chromatography systems. For instance, a
200 m2 bioreactor is the same height as a 10 m2 bioreactor, but
the diameter is different.
The scale-X carbo system is a single-use bioreactor coupled
with in-line product concentration operated by a bench-scale auto-
mated process controller (pH, DO, T, agitation, liquid flow rates),
which enables the production and simultaneous concentration of
viral products; a feature that is novel and differentiates this type
of fixed-bed bioreactor from others in the market. The fixed-bed
in the scale-X carbo bioreactor offers surface areas for cell growth
between 10 and 30 m2 (the growth area is equivalent to 120–360
roller bottles, 59–175 HYPERFlasksÒ, 16–48 CellSTACKÒ-10 layer,
or 6–16 HYPERStackÒ-36 layer vessels) in a total vessel volume
of 1.6–3.2 L, dependent on the surface area. This results in a high
cell density per unit volume and a compact footprint allowing inte-
gration in a standard biosafety cabinet.
Many commercially available fixed-bed bioreactors use ran-
domly packed disks or fabric strips as the substrate for cell attach-

https://doi.org/10.1016/j.vaccine.2020.03.041
0264-410X/Ó 2020 The Author(s). Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
3640 D.M. Berrie et al. / Vaccine 38 (2020) 3639–3645
ment; however, the scale-X bioreactor utilizes a fixed-bed that is
organized with a mesh layer which provides uniformity and
vessel-to-vessel consistency for cell growth. The fixed-bed consists
of a spiral winding that consists of alternating 5 cm2 wide ribbons
of a rigid polypropylene mesh for structure and media flow, and a
non-woven hydrophilized polyethylene terephthalate (PET) fabric
for cell entrapment and adhesion. This allows for homogeneous
radial and vertical cell distribution and linear media flow through
the mesh layer, which ensures homogeneous nutrient supply. Sam-
pling ports installed within the bioreactor lid allow for monitoring
of cell growth during culture through a manual process. This fea-
ture provides a quick and convenient access point for obtaining
samples for cell counting, ameliorating the risk of contamination
within the vessel. Environmental control is provided via a con-
trolled gas mixture to support cell growth and is supplied to the
headspace of the bioreactor (oxygen supply, CO2 stripping) with
allows for uptake through the falling film liquid–gas interface.
Another differentiator of this bioreactor focuses around an in-
line hollow-fiber filter, which can be utilized during the production
phase or at the time of harvest to concentrate produced product.
The virus-containing media is perfused out of the bioreactor vessel
into a dedicated ‘‘harvest” retentate container, while the media
within the bioreactor is replaced with fresh medium. This results
in a concentrated, low-volume bulk harvest from the bioreactor,
which allows for simplified and downsized downstream opera-
tions. In the present studies, we have added an in-line clarification
filter prior to the harvest bottle to mitigate fouling of the hollow-
fiber filter. This system provides all upstream and the initial down-
stream processes in a single standard sized biosafety cabinet.
Due to the convenient ergonomics of the scale-X carbo bioreac-
tor, we elected to utilize this system for development of a scalable
process to produce a recombinant Vesicular stomatitis virus
(rVSV)-based vaccine for Lassa virus (LASV). VSV is a negative-
sense, single stranded RNA virus that causes self-limited disease
in various livestock [8,9], and infection in humans results in a mild
flu-like syndrome or remains asymptomatic [9,10]. VSV is an
enveloped, bullet shaped virus approximately 70 nm
200 nm
[11]. Large foreign transgenes can be packaged and expressed
within VSV, making this virus an ideal candidate as a live-viral vac-
cine. A number of vectors expressing the glycoproteins from Ebola
virus (EBOV), Marburg virus (MARV), and LASV have previously
been constructed [12–16]. However, the scale required for produc-
ing material can be a limiting factor especially when an adherent
cell line is used as the producer. In this report, we demonstrate
the use of the Univercells scale-X bioreactor to produce rVSV-
LASV (VSVDG/LASVGP). When compared to classical flatstock pro-
duction, production from the scale-X carbo bioreactor resulted in a
4-log increase in virus production. Metabolite data was also mea-
sured; glutamine, ammonium, glucose, and lactate trends were
all consistent with normal cell growth in the bioreactor prior to
virus infection. Altogether, the data demonstrate that the scale-X
carbo bioreactor leads to higher production of virus per surface
area when compared to classical flatstock production, which
impacts the number of doses that can be produced per campaign.
In all, we describe a novel scalable fixed-bed bioreactor system
capable of producing high viral vaccine yields in a low environ-
mental footprint.
2. Material and methods
2.1. Fixed-bed bioreactor culture system
The 10 m2 Scale-X Carbo bioreactor (Univercells, Brussels, Bel-
gium) and in-line tangential flow filtration (TFF) module were used
for all experiments (Fig. 1). The bioreactor was inoculated at a tar-
get seeding density between 1.0 and 2.0
104 cells/cm2 (per man-
ufacturer’s instructions), and process parameters during the cell
expansion phase were set according to the manufacturer’s proto-
cols. Culture parameters were defined as temperature between
35 and 37 °C, pH between 7.0 and 7.6, agitation was set to 250
RPM, with a working volume of 1.6L. An external heated media
recirculation loop was connected to the bioreactor to support the
high cell density. The volume of media within the loop was large
enough to support a media to surface area ratio between 0.2 and
0.4 mL/cm2. The recirculation rate supported 20–30 bioreactor vol-
umes a day. The tubing manifolds on the reactor are greater than
¼” inner diameter, except the base feed line which is 1/800 inner
diameter.
2.2. Cell expansion
A Vero cell line (Ology Bioservices, Inc.) was cultured in a pro-
prietary serum-free media (Ology Bioservices Inc.). Cells were
expanded using flat stock and cell factories (Corning, Tewksbury,
MA) at a seeding density between 1.0 and 1.5
104 cells/cm2. Ves-
sels were cultured at 37 °C, 5% CO2. Cells were passaged until a
population of approximately 1.0–2.0
109 total viable cells was
achieved. At harvest, cell monolayers were washed with 1X Dul-
becco’s Phosphate-Buffered Saline, DPBS, (Gibco, Waltham, MA)
to remove excess spent media followed by dissociation with Try-
pLE CTS Select (Gibco, Waltham, MA). Centrifugation was per-
formed to remove TrypLE, and the cells were resuspended in
fresh medium. Cell counts were performed on a Vi-CELLTM XR Cell
Viability Analyzer (Beckman Coulter, Brea, California).
2.3. Cell density and metabolite analysis monitoring
Single-use sampling strips, inserted in the fixed-bed, were
removed daily for cell density determination utilizing lysis buffer
and nuclei counts. Sampling strips were lysed for 5 min followed
by vortexing for 1 min. Nuclei were stained with crystal violet to
visualize intact nuclei. Metabolite concentrations were measured
daily using the BioProfile Ò FLEX2 (Nova Biomedical, Waltham,
MA) by removing media samples from the aseptic sampling port.
A pH offset was performed when offline measures deviated
>±0.05 pH units from the online probe reading.
2.4. Infection process
Recombinant Vesicular stomatitis virus (rVSV) (minus the gly-
coprotein G) containing the Lassa virus (LASV) Josiah glycoprotein
(VSVDG/LASVGP) was graciously provided by the NIAID under
Material Transfer Agreement (LAB-18-P_LV-22 for in vitro use only
and for training and research purposes only). The stock VSVDG/
LASVGP, stock titer of 4.9
108 pfu/mL, was used for all infection
studies utilizing the bioreactor. Infection of the bioreactor with
virus inoculum was performed five days post-seeding or when
peak cell density was obtained, evident by nitrogen source deple-
tion as measured by glutamine. Briefly, the bioreactor was drained
of spent media and then refilled with fresh media containing the
viral inoculum. The recirculation loop was disconnected at the
point of infection, to perform a batch mode infection. Each run
was infected at a MOI of 0.05, and the infection process proceeded
for 48–72 h. Harvest was initiated once cell counts were depleted
on the sample strips. Infection of the flatstock vessels occurred at
the same time as the bioreactor, utilizing the same viral infection
inoculum.
 D.M. Berrie et al. / Vaccine 38 (2020) 3639–3645 3641

Fig. 1. Scale-X carbo Bioreactor. Representation of the scale-X carbo system featuring a 30 m2 bioreactor (red) located in the middle of the bioreactor control skid. A 5 L
harvest bottle is located on the right hand side of the skid with the inline TFF cartridge located behind the harvest bottle. Located on the left hand side of the control skid is a
control panel allowing the user to prime and pause the bioreactor pumps. Additionally, the inoculum and base bottles are shown to the left of the bioreactor. In the
experiments described, the 10 m2 was used, which has one-third of the height of the 30 m2 bioreactor. The external recirculation loop is not shown.

2.5. Viral harvest

Bulk harvest was passed through a two-step depth filtration

chain, Sartopure PP3 8 mm followed by a Sartopore 2 0.8/0.45 mm
filter, and collected into a secondary reservoir. In Run 4, after initial
emptying of the vessel, the bioreactor was rinsed with an addi-
tional 1 L of fresh medium to flush any residual virus particles con-
tained within the fixed-bed. This wash was passed through the
depth filters, and the TFF step was initiated for viral concentration.
The bulk harvest was concentrated 2-fold using a 100 kDa hallow-
fiber TFF cartridge. Flatstock vessels were harvested at the same
time as the bioreactor, with the bulk harvest clarified via centrifu-
gation at 1000g for 10 min.

2.6. Plaque assay

Samples of the bulk harvest collected for viral titer analyses via
plaque assay quantification using methods optimized by Ology
Bioservices, Inc. Briefly, a 10-fold dilution series was generated
and used to infect Vero cell monolayers. Infection proceeded for
2 days and the plates were then stained with crystal violet for
15 min, washed, and plaques were counted.

3. Results

3.1. Vero cell growth in the scale-X carbo system

The unique design of the scale-X carbo system allows for fluid
flow within the fixed bed and return via an internal centralized
channel to the impeller creating a continuous falling film mecha-
nism (Fig. 2). This falling film mechanism allows for gas exchange
to occur within the overlay of the bioreactor. A process establish-
ment run (Run 1) was performed using guidelines and process
parameters provided by the manufacturer. This run determined
that the Vero cell line would grow to consistent cell densities
within the 10 m2 fixed bed bioreactor (Table 1). The proceeding
runs were designed to establish various operating conditions to
achieve optimal cell density (Fig. 3). As demonstrated in Fig. 4, con-
sistent cell growth was observed in all runs from day 1 through day
5 after cell inoculation (day 0). During Run 3, the cells were
allowed to grow for one more day to determine whether that extra
Fig. 2. Scheme of the carbo bioreactor. The double layer nonwoven mesh (in blue)
surrounded by the woven mesh matrix is highlighted. Additionally, the fluid flow
directions are shown to indicate the flow of media throughout the bioreactor.
Probes for inline pH and DO monitoring are visible in the middle of the bioreactor.
day led to a significant growth of cells. It was concluded that wait-
ing until day 6 for infection did not lead to an overall higher cell
density that resulted in maximized virus production. Thus, it was
concluded that optimal cell growth occurred by day 5 post-
inoculation and that day 5 would be the trigger for future virus
production runs involving these Vero cells for virus infection.
3642 D.M. Berrie et al. / Vaccine 38 (2020) 3639–3645

Table 1 A few of the virus production parameters for campaigns 2 and 3

Scale-X carbo Cell Data. differed from campaign 1. First, the virus harvest was performed 3-
Scale-X Carbo Campaign Average Cell Data days post-infection, and second, the bulk harvest was filtered
Days Post Inoculation Cells/cm2 Total Cells (10.0 m2) through a 2-step depth filtration chain to remove excess cell debris
prior to collection in the retentate vessel. Negligible virus was
1 1.81E+04 1.81E+09
2 3.51E+04 3.51E+09
retained by the depth filters post-harvest (data not shown) and
3 5.88E+04 5.88E+09 overall production of virus was similar to Campaign 1 for each of
4 9.96E+04 9.96E+09 these campaigns. The average bulk virus titers from campaigns 2
5 1.11E+05 1.11E+10 and 3 were 5.03
1012 pfu/mL and 2.90
1012 pfu/mL, respec-
Day of Infection 1.51E+05 1.51E+10
tively, correlating to a surface area normalized harvest of
1 Day Post-Infection 5.69E+04 5.69E+09
Day of Harvest 2.93E+03 2.93E+08 7.55
1010 pfu/cm2 and 4.35
1010 pfu/cm2. No statistical differ-
ence of the titers was calculated between these two runs and
Cells were counted by collecting the sampling strip, lysing the attached cells, and
among the first three campaigns, overall.
counting nuclei at various time points as described. The cell counts presented are
the averages of four distinct bioreactor runs. The final campaign (Run #4) involved the in-line tangential
flow filtration (TFF) step for virus concentration after the bulk har-
vest was subjected to clarification via depth filtration. A schematic
3.2. Metabolic trending of the in-line TFF, the bioreactor, and the retentate reservoir is
demonstrated in Fig. 5. All of the cell inoculation and infection
During the cell growth phase, key metabolic activity was mea- parameters followed the procedures performed in campaigns 2
sured including the consumption of glutamine followed by glucose, and 3. However, after the bulk virus was collected, the supernatant
and subsequent production of ammonium and lactate (Fig. 4). Glu- material was subjected to TFF for concentration. This TFF process
tamine concentration at cell inoculation was 2.4 ± 0.2 mmol/L and was not performed on the previous campaigns. The addition of
was consumed below the linear range of the assay. Similarly, glu- the TFF step allowed for an extra washing of the bioreactor to col-
cose concentration was 17.57 ± 1.27 mmol/L at inoculation and lect any residual virus that could be located within the bedding of
was consumed below the linear range of the assay. The maximum the bioreactor. The viral harvest was concentrated from 1.6 L to
lactate production observed was 16.71 mmol/L and maximum 750 mL, and the titer of the bulk harvested was calculated at
observed ammonium ions concentration was 2.77 mmol/L. 1.03
1012 pfu/mL (Table 2). After the TFF concentration step
was completed, a sample of the retentate was titered at
3.3. Production of VSVDG/LASVGP 2.47
1012 pfu/mL (Table 2). This titer is aligned with the 2x con-
centration step, and the excess virus could be associated to virus
During the first virus production campaign (Run 1), VSVDG/ recovery from the bedding after washing or is a result of the natu-
LASVGP infection was initiated 5 days post-bioreactor inoculation ral variation associated with the plaque assay.
at a MOI of 0.05. Following infection, significant cell debris was Lastly, when compared to production in a T-225 flask or
observed in the supernatant within the bioreactor 2 days post- CellSTACK-10, a significant increase in the amount of virus produc-
infection triggering the harvest of virus. The bioreactor was tion was observed when utilizing the scale-X bioreactor. An
drained to collect the virus-containing supernatant, and a media increase of over 4 logs of virus per mL was observed, regardless
rinse step was performed to flush any residual virus from the of the parameters associated with each independent bioreactor
holdup volume. Once collection of the VSVDG/LASVGP was com- run. This correlates to an increase of 4 to 7 logs of virus per biore-
pleted, the virus was titered using a standard plaque assay. A titer actor when comparing to production from the CellSTACK-10 and T-
of 4.25
1012 pfu/mL was calculated, which corresponds to a total 225. Overall, the results of the scale-X carbo runs confirm the use
titer of 6.80
1015 pfu per this campaign and a normalized titer of of this bioreactor as a means to produce, clarify, and concentrate
6.80
1010 pfu/cm2 (Table 2). live virus.

Fig. 3. Cell growth kinetics from individual bioreactor campaigns. Cell counts are shown up to the time of infection of the bioreactor. Different campaigns are denoted as
follows: campaign 1 ( ), campaign 2 ( ), campaign 3 ( ), and campaign 4 ( ).
 D.M. Berrie et al. / Vaccine 38 (2020) 3639–3645 3643

Fig. 4. Metabolites concentration trends for Vero cell growth in 10 L carbo bioreactor, by campaign. Metabolites, associated with cell growth, glutamine (A), ammonium (B),
glucose (C), and lactate (D) were analyzed prior to infection. Different campaigns are denoted as follows: campaign 1 ( ), campaign 2 ( ), campaign 3 ( ), and campaign 4
( ).

Table 2
Comparison of Viral Titers.

Scale-X Carbo Campaigns

Production System Surface Area Titer (pfu/mL) Total harvest (mL) Total Virus (pfu) Virus Production (pfu/cm2)
T225 225 cm2 1.10
107 60 6.00
108 2.67
106
CellSTACK 10 6360 cm2 8.66
107 1500 1.30
1011 5.77
108
Scale-X Carbo Run 1 10 m2 4.25
1012 1600 6.80
1015 6.80
1010
Scale-X Carbo Run 2 10 m2 5.03
1012 1600 7.55
1015 7.55
1010
Scale-X Carbo Run 3 10 m2 2.90
1012 1600 4.35
1015 4.35
1010
Scale-X Carbo Run 4 10 m2 1.03
1012 1600 1.65
1015 1.65
1010
Scale-X Carbo Run 4 (Retentate) NA 2.47
1012 750 1.85
1015 NA

Comparison of viral titers as a result of virus production from different vessels as well as the individual bioreactor campaigns. The result from in-line viral concentration
during Run 4 is also presented.

4. Discussion to limit the impact of scale-up processes. Additionally, the matrix

Single use technologies continue to be the gold standard in bio-
logics manufacturing with scalability limiting the use of adherent
cell fixed-bed bioreactor systems. The scale-X line of bioreactors
ranges from 2.4 m2 benchtop systems through the commercial
scale 600 m2 system. The scale-X carbo bioreactor has proven to
be a useful tool for bridging the scalability gap found in other
fixed-bed bioreactor systems. Similar systems do not have the sur-
face area options for intermediate scales such as 10 m2 or 30 m2.
This scale is useful for generating material for a wide range of uses
such as preclinical toxicology lots, development of downstream
methods, analytical methods, generation of reference material,
and phase I clinical studies. A limitation found with other fixed-
bed bioreactor systems revolves around a decrease in production
efficiency when scale-up to larger fixed bed diameter occurs. The
scale-X carbo line increases bed height while maintaining diameter
of the fixed-bed is another unique feature of this bioreactor that
differentiates it from other fixed-bed bioreactor systems. The
fixed-bed consists of alternating 5 cm2 wide ribbons of non-
woven PET fabric and a polypropylene mesh layer that are spiral
wound. The PET fabric provides a 3-dimensional micro-
environment for cell attachment and growth; the polypropylene
mesh provides structure and a channel for fluid flow. The spiral
design of the fixed bed along with the increase in the height of
the bioreactor allow for even radial and vertical cell distribution
within the bioreactor, which can be monitored by collecting strips
of PET fabric inserted in the fixed-bed that can be accessed through
sampling ports within the lid of the bioreactor system. These eight
access ports are evenly distributed around the lid, and they are
characterized by a locking port system that contains a cap magnet-
ically connected to plastic rods that connect to the PET strips. The
cap and rod system is easily removed from the bioreactor and the
3644 D.M. Berrie et al. / Vaccine 38 (2020) 3639–3645
Fig. 5. Schematic of the in-line purification strategy. The scale-X carbo bioreactor is
connected to the in-line TFF cartridge and harvest container. In some cases, a depth
filter train, not shown, was added the downstream processing components to the
exit line from the bioreactor prior to the harvest bottle.
magnetic capability of the rod and cap allow for easy removal of
the rod from the cap and the replacement of the cap back to the
access port while maintaining aseptic practices. The strips can then
be removed from the rod, and PET strips can be used to determine
the cell count per surface after lysing of the cells and counting of
nuclei. The location of the access ports and the distribution of
the PET strips throughout the fixed-bed allows for the determina-
tion of cell density and distribution.
Moreover, the PET-polypropylene mesh spiral technology
allows for a homogenous nutrient supply due to the pattern of
media flow through the polypropylene mesh layers of the spiral
fixed-bed (Fig. 2). The media recirculation flow rate has been set
to achieve approximately 25 media changes per day. The
exchanges are required for both the supply of fresh nutrients as
well as the removal of toxic metabolites prior to their buildup,
and the results presented demonstrate consistent cell growth dur-
ing the cell expansion stage within the bioreactor. Additional
exchanges could be performed if the metabolic requirements of
the cells dictated the need for increased recirculation.
Lastly, the bioreactor features an in-line TFF hollow-fiber sys-
tem that allows for the purification and concentration of the virus
product in a continuous and aseptic manner. For clarification pro-
cesses, and to mitigate the fouling of the hollow-fiber system due
to cell debris that flows from the bioreactor during harvest, a depth
filter train was welded to the tubing system that exits the bioreac-
tor. This allows for an additional clarification step and the capture
of cell debris during the in-line purification process. This is essen-
tial when propagating virus that cause cell lysis within the bioreac-
tor and purifying viral product through the in-line TFF system.
Our aim for this campaign was to first determine if a Vero cell
line would propagate after binding to the bioreactor PET fabric
bedding, and secondly, determine how production of a viral vac-
cine candidate within the bioreactor compares to traditional flat-
stock production. Cell densities within the bioreactor were higher
than maximum cell densities observed in T-225 flasks or
CellSTACK-10 vessels when normalizing cell counts to an equal
surface area. This translates into the potential for higher virus pro-
ductivity simply due to number of cells per surface area, and not
taking into account the overall fitness of the cells, which could lead
to higher virus production. Additionally, the metabolite profile of
the cells growing in the bioreactor was consistent over the four
runs and indicative of overall cell fitness. As for virus production,
it is difficult to compare the titer based on the volume of the ves-
sels and bioreactor since a different volume is used for each. For
instance, the volume of the harvest from a T-225 vessel is approx-
imately 60 to 65 mL, while the volume harvested from a
CellSTACK-10 and scale-X carbo is approximately 1.5 L and 1.8 L,
respectively. As noted in Table 2, the titers are reported per vol-
ume, in addition to the total harvest. However, to normalize pro-
duction for comparison, the titers are reported per surface area.
This allows for a more complete comparison of virus production
in each of the different vessels and bioreactors. On average, a T-
225 yielded 2.67
106 pfu/cm2, while a CellSTACK-10 yielded
approximately 5.77
108 pfu/cm2. The average titer from the four
distinct scale-X carbo runs was approximately 5.09
1010 pfu/
cm2. After normalization per surface area, the scale-X carbo biore-
actor yielded an increase of approximately 2 logs of virus when
compared to the CellSTACK-10 and over 4 logs of virus when com-
pared to the T-225. Furthermore, the high titers that result from
scale-X carbo production occur through a small volume of media
measuring approximately 1.6 L. This small volume allows for less
complicated downstream procedures associated with clarification,
purification, and concentration. The addition of the depth filters for
clarification and the in-line TFF for purification and concentration
allows for a continuous aseptic downstream process resulting in
the collection of viral product in the retentate reservoir. The down-
stream purification process is normally associated with some
degree of virus product loss. However, use of the in-line TFF
hollow-fiber concentration system did not appear to result in the
loss of viral product. This may be explained by washing of the
bioreactor during harvest to remove any residual virus that may
have been trapped within the fixed-bedding matrix, the addition
of the depth filter for clarification of the cell debris, and the proper
sizing of the filters. The in-line TFF allows for a washing step after
virus harvest to collect any residual virus since the TFF allows for 2
to 3x concentration, which negates the extra volume as a result of
washing. Lastly, due to the various sizes of different viruses, it
should be mentioned the filters need to be properly chosen accord-
ing to size to avoid loss of the virus in the filter during purification.
In conclusion, the increase in viral vaccine production is likely
due to multiple factors focusing around the number of cells per
surface area and the general fitness of the cells, which may be
due to the geometry of the PET fiber bedding providing for cell den-
sity uniformity, entrapment, and adhesion. Ultimately, these fac-
tors lead to a homogenous nutrient supply from media that flows
through the polypropylene mesh layer of the fixed-bed. This sys-
tem addresses a critical need for low cost, high yield production
of vaccines and represents another step forward for large scale pro-
duction of live-virus vaccines. Ultimately, the increase in titers per
surface area may lead to an increase in the number of dosages per
campaign.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
The authors wish to thank Isabel Scholtz for her thoughtful discus-
sions over syntax and descriptions of the assays. Additionally, the
authors thank NIAID for generously providing the rVSV-LASV used
in this study.
Authors note
DB, RW, CM, and EV are all employees of Ology Bioservices Inc.
AC is an employee of Univercells.
 D.M. Berrie et al. / Vaccine 38 (2020) 3639–3645
Funding
Funding for this study was provided by Ology Bioservices Inc.
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