Development of a high-yield live-virus vaccine production platform … · 2020. 4. 2. · Development of a high-yield live-virus vaccine production platform using a novel ﬁxed-bed

Vaccine xxx (xxxx) xxx

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Vaccine

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Development of a high-yield live-virus vaccine production platformusing a novel fixed-bed bioreactor

https://doi.org/10.1016/j.vaccine.2020.03.0410264-410X/� 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/).

⇑ Corresponding author.E-mail address: [email protected] (E.M. Vela).

Please cite this article as: D. M. Berrie, R. C. Waters, C. Montoya et al., Development of a high-yield live-virus vaccine production platform usingfixed-bed bioreactor, Vaccine, https://doi.org/10.1016/j.vaccine.2020.03.041

Dalton M. Berrie a, Robin C. Waters a, Christopher Montoya a, Alex Chatel b, Eric M. Vela a,⇑aOlogy Bioservices, Process Development, 13200 NW Nano Ct., Alachua, FL 32615, USAbUnivercells, Rue de la Maîtise 11, 1400 Nivelles, Belgium

a r t i c l e i n f o

Article history:Received 16 December 2019Received in revised form 17 March 2020Accepted 22 March 2020Available online xxxx

Keywords:Vesicular stomatitis virusFixed-bed bioreactorVaccine developmentscale-X carbo

a b s t r a c t

The increasing importance of viral vaccine manufacturing has driven the need for high cell density pro-cess optimization that allows for higher production levels. Vero cells are one of the more popular adher-ent cell lines used for viral vaccine production. However, production is limited due to the logisticallimitations surrounding adherent cell line processes, such as large equipment footprints, time andlabor-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.The unit is compact and is scalable and one of the novel features of the system is the continuous in-line downstream purification and concentration processes associated with the bioreactor vessel. We pre-sent the results from a campaign featuring a proprietary Vero cell line for production of a live recombi-nant Vesicular stomatitis virus vaccine that features the Lassa Fever virus glycoproteins. Metaboliteanalyses 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 cellfactories production, 5.77 � 108 pfu/cm2. Overall, we describe a novel bioreactor platform that allows fora cost-efficient and scalable process for viral vaccine production.� 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 foremploying adherent cells for production of a myriad of variousviral vectors, live viruses, and virus-based vaccines. These bioreac-tors combine a low shear environment of flatware systems withautomation and scalability. The Pall iCELLis� and EppendorfFibra-Cel� systems have been used to produce high yields of viralvectors and live viruses [1–4]. There are significant advantagesfor utilizing these closed fixed-bed bioreactor systems, whichinclude 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 themarket is the scale-XTM bioreactor system from Univercells. Thescale-X portfolio offers a range of growth surfaces: scale-X ‘hydro’(<3 m2), ‘carbo’ (10–30 m2), and ‘nitro’ (200–600 m2). This rangeoffers a scalable process and the capability for clinical lot produc-tion. Within the Univercells product line, the bioreactor height

increases, while the diameter is held constant. For example, thecarbo 10 m2 bioreactor is 1/3 the height of a 30 m2 bioreactor.However, scale-up among the different lines is achieved by keepingthe height of the fixed-bed constant and increasing the diameter,similar to scale-up in chromatography systems. For instance, a200 m2 bioreactor is the same height as a 10 m2 bioreactor, butthe diameter is different.

The scale-X carbo system is a single-use bioreactor coupledwith 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 ofviral products; a feature that is novel and differentiates this typeof fixed-bed bioreactor from others in the market. The fixed-bedin the scale-X carbo bioreactor offers surface areas for cell growthbetween 10 and 30 m2 (the growth area is equivalent to 120–360roller bottles, 59–175 HYPERFlasks�, 16–48 CellSTACK�-10 layer,or 6–16 HYPERStack�-36 layer vessels) in a total vessel volumeof 1.6–3.2 L, dependent on the surface area. This results in a highcell 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-

a novel

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ment; however, the scale-X bioreactor utilizes a fixed-bed that isorganized with a mesh layer which provides uniformity andvessel-to-vessel consistency for cell growth. The fixed-bed consistsof a spiral winding that consists of alternating 5 cm2 wide ribbonsof a rigid polypropylene mesh for structure and media flow, and anon-woven hydrophilized polyethylene terephthalate (PET) fabricfor cell entrapment and adhesion. This allows for homogeneousradial and vertical cell distribution and linear media flow throughthe mesh layer, which ensures homogeneous nutrient supply. Sam-pling ports installed within the bioreactor lid allow for monitoringof cell growth during culture through a manual process. This fea-ture provides a quick and convenient access point for obtainingsamples for cell counting, ameliorating the risk of contaminationwithin the vessel. Environmental control is provided via a con-trolled gas mixture to support cell growth and is supplied to theheadspace of the bioreactor (oxygen supply, CO2 stripping) withallows 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 productionphase or at the time of harvest to concentrate produced product.The virus-containing media is perfused out of the bioreactor vesselinto a dedicated ‘‘harvest” retentate container, while the mediawithin the bioreactor is replaced with fresh medium. This resultsin 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 clarificationfilter 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 scalableprocess 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 diseasein various livestock [8,9], and infection in humans results in a mildflu-like syndrome or remains asymptomatic [9,10]. VSV is anenveloped, bullet shaped virus approximately 70 nm � 200 nm[11]. Large foreign transgenes can be packaged and expressedwithin VSV, making this virus an ideal candidate as a live-viral vac-cine. A number of vectors expressing the glycoproteins from Ebolavirus (EBOV), Marburg virus (MARV), and LASV have previouslybeen constructed [12–16]. However, the scale required for produc-ing material can be a limiting factor especially when an adherentcell line is used as the producer. In this report, we demonstratethe 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 a4-log increase in virus production. Metabolite data was also mea-sured; glutamine, ammonium, glucose, and lactate trends wereall consistent with normal cell growth in the bioreactor prior tovirus infection. Altogether, the data demonstrate that the scale-Xcarbo bioreactor leads to higher production of virus per surfacearea when compared to classical flatstock production, whichimpacts the number of doses that can be produced per campaign.In all, we describe a novel scalable fixed-bed bioreactor systemcapable 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 usedfor all experiments (Fig. 1). The bioreactor was inoculated at a tar-

Please cite this article as: D. M. Berrie, R. C. Waters, C. Montoya et al., Developfixed-bed bioreactor, Vaccine, https://doi.org/10.1016/j.vaccine.2020.03.041

get seeding density between 1.0 and 2.0 � 104 cells/cm2 (per man-ufacturer’s instructions), and process parameters during the cellexpansion phase were set according to the manufacturer’s proto-cols. Culture parameters were defined as temperature between35 and 37 �C, pH between 7.0 and 7.6, agitation was set to 250RPM, with a working volume of 1.6L. An external heated mediarecirculation loop was connected to the bioreactor to support thehigh cell density. The volume of media within the loop was largeenough to support a media to surface area ratio between 0.2 and0.4 mL/cm2. The recirculation rate supported 20–30 bioreactor vol-umes a day. The tubing manifolds on the reactor are greater than¼” inner diameter, except the base feed line which is 1/800 innerdiameter.

2.2. Cell expansion

A Vero cell line (Ology Bioservices, Inc.) was cultured in a pro-prietary serum-free media (Ology Bioservices Inc.). Cells wereexpanded 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 apopulation of approximately 1.0–2.0 � 109 total viable cells wasachieved. 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 infresh medium. Cell counts were performed on a Vi-CELLTM XR CellViability Analyzer (Beckman Coulter, Brea, California).

2.3. Cell density and metabolite analysis monitoring

Single-use sampling strips, inserted in the fixed-bed, wereremoved daily for cell density determination utilizing lysis bufferand nuclei counts. Sampling strips were lysed for 5 min followedby vortexing for 1 min. Nuclei were stained with crystal violet tovisualize intact nuclei. Metabolite concentrations were measureddaily using the BioProfile � 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 underMaterial Transfer Agreement (LAB-18-P_LV-22 for in vitro use onlyand for training and research purposes only). The stock VSVDG/LASVGP, stock titer of 4.9 � 108 pfu/mL, was used for all infectionstudies utilizing the bioreactor. Infection of the bioreactor withvirus inoculum was performed five days post-seeding or whenpeak cell density was obtained, evident by nitrogen source deple-tion as measured by glutamine. Briefly, the bioreactor was drainedof spent media and then refilled with fresh media containing theviral inoculum. The recirculation loop was disconnected at thepoint of infection, to perform a batch mode infection. Each runwas infected at a MOI of 0.05, and the infection process proceededfor 48–72 h. Harvest was initiated once cell counts were depletedon the sample strips. Infection of the flatstock vessels occurred atthe same time as the bioreactor, utilizing the same viral infectioninoculum.

ment of a high-yield live-virus vaccine production platform using a novel


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 Lharvest 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 acontrol 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 theexperiments 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.

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 flowdirections 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.

D.M. Berrie et al. / Vaccine xxx (xxxx) xxx 3

2.5. Viral harvest

Bulk harvest was passed through a two-step depth filtrationchain, Sartopure PP3 8 mm followed by a Sartopore 2 0.8/0.45 mmfilter, and collected into a secondary reservoir. In Run 4, after initialemptying 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 thedepth 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 sametime 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 viaplaque assay quantification using methods optimized by OlogyBioservices, Inc. Briefly, a 10-fold dilution series was generatedand used to infect Vero cell monolayers. Infection proceeded for2 days and the plates were then stained with crystal violet for15 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 fluidflow within the fixed bed and return via an internal centralizedchannel to the impeller creating a continuous falling film mecha-nism (Fig. 2). This falling film mechanism allows for gas exchangeto occur within the overlay of the bioreactor. A process establish-ment run (Run 1) was performed using guidelines and processparameters provided by the manufacturer. This run determinedthat the Vero cell line would grow to consistent cell densitieswithin the 10 m2 fixed bed bioreactor (Table 1). The proceedingruns were designed to establish various operating conditions toachieve optimal cell density (Fig. 3). As demonstrated in Fig. 4, con-sistent cell growth was observed in all runs from day 1 through day5 after cell inoculation (day 0). During Run 3, the cells wereallowed to grow for one more day to determine whether that extra


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 celldensity that resulted in maximized virus production. Thus, it wasconcluded that optimal cell growth occurred by day 5 post-inoculation and that day 5 would be the trigger for future virusproduction runs involving these Vero cells for virus infection.



Table 1Scale-X carbo Cell Data.

Scale-X Carbo Campaign Average Cell Data

Days Post Inoculation Cells/cm2 Total Cells (10.0 m2)

1 1.81E+04 1.81E+092 3.51E+04 3.51E+093 5.88E+04 5.88E+094 9.96E+04 9.96E+095 1.11E+05 1.11E+10Day of Infection 1.51E+05 1.51E+101 Day Post-Infection 5.69E+04 5.69E+09Day of Harvest 2.93E+03 2.93E+08

Cells were counted by collecting the sampling strip, lysing the attached cells, andcounting nuclei at various time points as described. The cell counts presented arethe averages of four distinct bioreactor runs.


3.2. Metabolic trending

During the cell growth phase, key metabolic activity was mea-sured including the consumption of glutamine followed by glucose,and subsequent production of ammonium and lactate (Fig. 4). Glu-tamine concentration at cell inoculation was 2.4 ± 0.2 mmol/L andwas consumed below the linear range of the assay. Similarly, glu-cose concentration was 17.57 ± 1.27 mmol/L at inoculation andwas consumed below the linear range of the assay. The maximumlactate production observed was 16.71 mmol/L and maximumobserved ammonium ions concentration was 2.77 mmol/L.

3.3. Production of VSVDG/LASVGP

During the first virus production campaign (Run 1), VSVDG/LASVGP infection was initiated 5 days post-bioreactor inoculationat a MOI of 0.05. Following infection, significant cell debris wasobserved in the supernatant within the bioreactor 2 days post-infection triggering the harvest of virus. The bioreactor wasdrained to collect the virus-containing supernatant, and a mediarinse step was performed to flush any residual virus from theholdup volume. Once collection of the VSVDG/LASVGP was com-pleted, the virus was titered using a standard plaque assay. A titerof 4.25 � 1012 pfu/mL was calculated, which corresponds to a totaltiter of 6.80 � 1015 pfu per this campaign and a normalized titer of6.80 � 1010 pfu/cm2 (Table 2).

Fig. 3. Cell growth kinetics from individual bioreactor campaigns. Cell counts are showfollows: campaign 1 ( ), campaign 2 ( ), campaign 3 ( ), and campaign 4 ( ).


A few of the virus production parameters for campaigns 2 and 3differed from campaign 1. First, the virus harvest was performed 3-days post-infection, and second, the bulk harvest was filteredthrough a 2-step depth filtration chain to remove excess cell debrisprior to collection in the retentate vessel. Negligible virus wasretained by the depth filters post-harvest (data not shown) andoverall production of virus was similar to Campaign 1 for each ofthese campaigns. The average bulk virus titers from campaigns 2and 3 were 5.03 � 1012 pfu/mL and 2.90 � 1012 pfu/mL, respec-tively, correlating to a surface area normalized harvest of7.55 � 1010 pfu/cm2 and 4.35 � 1010 pfu/cm2. No statistical differ-ence of the titers was calculated between these two runs andamong the first three campaigns, overall.

The final campaign (Run #4) involved the in-line tangentialflow filtration (TFF) step for virus concentration after the bulk har-vest was subjected to clarification via depth filtration. A schematicof the in-line TFF, the bioreactor, and the retentate reservoir isdemonstrated in Fig. 5. All of the cell inoculation and infectionparameters followed the procedures performed in campaigns 2and 3. However, after the bulk virus was collected, the supernatantmaterial was subjected to TFF for concentration. This TFF processwas not performed on the previous campaigns. The addition ofthe TFF step allowed for an extra washing of the bioreactor to col-lect any residual virus that could be located within the bedding ofthe bioreactor. The viral harvest was concentrated from 1.6 L to750 mL, and the titer of the bulk harvested was calculated at1.03 � 1012 pfu/mL (Table 2). After the TFF concentration stepwas completed, a sample of the retentate was titered at2.47 � 1012 pfu/mL (Table 2). This titer is aligned with the 2x con-centration step, and the excess virus could be associated to virusrecovery from the bedding after washing or is a result of the natu-ral variation associated with the plaque assay.

Lastly, when compared to production in a T-225 flask orCellSTACK-10, a significant increase in the amount of virus produc-tion was observed when utilizing the scale-X bioreactor. Anincrease of over 4 logs of virus per mL was observed, regardlessof the parameters associated with each independent bioreactorrun. This correlates to an increase of 4 to 7 logs of virus per biore-actor when comparing to production from the CellSTACK-10 and T-225. Overall, the results of the scale-X carbo runs confirm the useof this bioreactor as a means to produce, clarify, and concentratelive virus.

n up to the time of infection of the bioreactor. Different campaigns are denoted as



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 2Comparison 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 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 concentrationduring Run 4 is also presented.


4. Discussion

Single use technologies continue to be the gold standard in bio-logics manufacturing with scalability limiting the use of adherentcell fixed-bed bioreactor systems. The scale-X line of bioreactorsranges from 2.4 m2 benchtop systems through the commercialscale 600 m2 system. The scale-X carbo bioreactor has proven tobe a useful tool for bridging the scalability gap found in otherfixed-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 usessuch as preclinical toxicology lots, development of downstreammethods, 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 productionefficiency when scale-up to larger fixed bed diameter occurs. Thescale-X carbo line increases bed height while maintaining diameter


to limit the impact of scale-up processes. Additionally, the matrixof the fixed-bed is another unique feature of this bioreactor thatdifferentiates it from other fixed-bed bioreactor systems. Thefixed-bed consists of alternating 5 cm2 wide ribbons of non-woven PET fabric and a polypropylene mesh layer that are spiralwound. The PET fabric provides a 3-dimensional micro-environment for cell attachment and growth; the polypropylenemesh provides structure and a channel for fluid flow. The spiraldesign of the fixed bed along with the increase in the height ofthe bioreactor allow for even radial and vertical cell distributionwithin the bioreactor, which can be monitored by collecting stripsof PET fabric inserted in the fixed-bed that can be accessed throughsampling ports within the lid of the bioreactor system. These eightaccess ports are evenly distributed around the lid, and they arecharacterized by a locking port system that contains a cap magnet-ically connected to plastic rods that connect to the PET strips. Thecap and rod system is easily removed from the bioreactor and the



Fig. 5. Schematic of the in-line purification strategy. The scale-X carbo bioreactor isconnected to the in-line TFF cartridge and harvest container. In some cases, a depthfilter train, not shown, was added the downstream processing components to theexit line from the bioreactor prior to the harvest bottle.


magnetic capability of the rod and cap allow for easy removal ofthe rod from the cap and the replacement of the cap back to theaccess port while maintaining aseptic practices. The strips can thenbe removed from the rod, and PET strips can be used to determinethe cell count per surface after lysing of the cells and counting ofnuclei. The location of the access ports and the distribution ofthe PET strips throughout the fixed-bed allows for the determina-tion of cell density and distribution.

Moreover, the PET-polypropylene mesh spiral technologyallows for a homogenous nutrient supply due to the pattern ofmedia flow through the polypropylene mesh layers of the spiralfixed-bed (Fig. 2). The media recirculation flow rate has been setto achieve approximately 25 media changes per day. Theexchanges are required for both the supply of fresh nutrients aswell 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. Additionalexchanges could be performed if the metabolic requirements ofthe 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 virusproduct in a continuous and aseptic manner. For clarification pro-cesses, and to mitigate the fouling of the hollow-fiber system dueto cell debris that flows from the bioreactor during harvest, a depthfilter train was welded to the tubing system that exits the bioreac-tor. This allows for an additional clarification step and the captureof 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 cellline would propagate after binding to the bioreactor PET fabricbedding, 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 higherthan maximum cell densities observed in T-225 flasks orCellSTACK-10 vessels when normalizing cell counts to an equalsurface area. This translates into the potential for higher virus pro-ductivity simply due to number of cells per surface area, and nottaking into account the overall fitness of the cells, which could leadto higher virus production. Additionally, the metabolite profile ofthe cells growing in the bioreactor was consistent over the fourruns 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 aCellSTACK-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 productionin each of the different vessels and bioreactors. On average, a T-225 yielded 2.67 � 106 pfu/cm2, while a CellSTACK-10 yieldedapproximately 5.77 � 108 pfu/cm2. The average titer from the fourdistinct 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 whencompared to the CellSTACK-10 and over 4 logs of virus when com-pared to the T-225. Furthermore, the high titers that result fromscale-X carbo production occur through a small volume of mediameasuring approximately 1.6 L. This small volume allows for lesscomplicated downstream procedures associated with clarification,purification, and concentration. The addition of the depth filters forclarification and the in-line TFF for purification and concentrationallows for a continuous aseptic downstream process resulting inthe collection of viral product in the retentate reservoir. The down-stream purification process is normally associated with somedegree of virus product loss. However, use of the in-line TFFhollow-fiber concentration system did not appear to result in theloss of viral product. This may be explained by washing of thebioreactor during harvest to remove any residual virus that mayhave been trapped within the fixed-bedding matrix, the additionof the depth filter for clarification of the cell debris, and the propersizing of the filters. The in-line TFF allows for a washing step aftervirus harvest to collect any residual virus since the TFF allows for 2to 3x concentration, which negates the extra volume as a result ofwashing. Lastly, due to the various sizes of different viruses, itshould 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 likelydue to multiple factors focusing around the number of cells persurface area and the general fitness of the cells, which may bedue 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 flowsthrough the polypropylene mesh layer of the fixed-bed. This sys-tem addresses a critical need for low cost, high yield productionof vaccines and represents another step forward for large scale pro-duction of live-virus vaccines. Ultimately, the increase in titers persurface area may lead to an increase in the number of dosages percampaign.

Declaration of Competing Interest

The authors declare that they have no known competing finan-cial interests or personal relationships that could have appearedto 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, theauthors thank NIAID for generously providing the rVSV-LASV usedin this study.

Authors note

DB, RW, CM, and EV are all employees of Ology Bioservices Inc.AC is an employee of Univercells.




Funding

Funding for this study was provided by Ology Bioservices Inc.

References

[1] Lennaertz A, Knowles S, Drugmand J-C, Castillo J. Viral vector production in theintegrity� iCELLis�. BMC Proc 2013().

[2] Legmann R. Case study: single-use platform for complete process developmentand scale-up of an adenovirus. Pall Life Sciences.

[3] Meuwly F, Ruffieuyx P-A, Kadouri A, von Stockar U. Packed-bed bioreactors formammalian cell culture. Biotechnol. Adv. 2007:45–56.

[4] Merten OW, Cruz PE, Rochette C, Geny-Fiamma C, Bouquet C, Goncalves D,Danos O, Carrondo MJT. Comparison of different bioreactor systems for theproduction of. Biotechnol Prog 2001:326–35.

[5] Rajendran R, Lingala R, Kumar VS, Obulapathi BB, Manickam E, Rao MS, et al.Assessment of packed bed bioreactor systems in the production of viralvaccines. AMB Exp 2014:25–31.

[6] Tapia F, Vázquez-Ramírez D, Genzel Y, Reichl U. Bioreactors for high celldensity and continuous multi-stage cultivations: options for processintensification in cell culture-based viral vaccine production. Appl MicrobiolBiotechnol 2016:2121–32.

[7] Moroni L, Schrooten J, Truckenmuller R, Rouwkema J, Sohier J, van BlitterswijkCA. Chapter 1 - Tissue Engineering: An Introduction. Academic Press; 2014.

[8] Lichty Brian D, Power Anthony T, Stojdl David F, Bell John C. Vesicularstomatitis virus: re-inventing the bullet. Trends Mol Med 2004;10(5):210–6. ,


https://linkinghub.elsevier.com/retrieve/pii/S1471491404000760. https://doi.org/10.1016/j.molmed.2004.03.003.

[9] Fine SM. Vesicular stomatitis virus and related vesiculoviruses, 8th ed., vol. 2;2015, p. 1981–83.

[10] Strauss JH, Strauss EG. Minus-strand RNA viruses. Virs Human Dis2008:137–91.

[11] Cureton DK, Massol RH, Whelan SPJ, Kirchhausen T. The length of vesicularstomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis. PLoS Pathog 2010;6(9).

[12] Henao-Restrepo AM, Camacho A, et al. Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from theGuinea ring vaccination, open-label, cluster-randomised trial (Ebola ÇaSuffit!). Lancet 2017:505–18.

[13] Geisbert T, Jones S, Fritz E, Shurtleff A, Geisbert J, et al. Development of a newvaccine for the prevention of lassa fever. PLoS Med 2005.

[14] Monath T, Fast P, Modjarrad K, Clarke D, Martin D, Fusco J, Nichols R, et al.rVSVdG-ZEBOV-GP (also designated V920) recombinant vesicular stomatitisvirus pseudotyped with Ebola Zaire Glycoprotein: Standardized template withkey considerations for a risk/benefit assessment, vol. 1; 2019.

[15] Velazquez-Salinas L, Naik S, Pauszek S, Peng KW, Russell S, Rodriguez L.Oncolytic recombinant vesicular stomatitis virus (VSV) is nonpathogenic andnontransmissible in pigs, a natural host of VSV. Hum Gene Ther Clin Dev2017;28(2):108–15.

[16] Muik Alexander, Dold Catherine, Geiß Yvonne, Volk Andreas, Werbizki Marina,Dietrich Ursula, von Laer Dorothee. Semireplication-competent vesicularstomatitis virus as a novel platform for oncolytic virotherapy. J Mol Med2012;90(8):959–70. http://link.springer.com/10.1007/s00109-012-0863-6.https://doi.org/10.1007/s00109-012-0863-6.


http://refhub.elsevier.com/S0264-410X(20)30420-5/h0005


















https://linkinghub.elsevier.com/retrieve/pii/S1471491404000760

https://doi.org/10.1016/j.molmed.2004.03.003

https://doi.org/10.1016/j.molmed.2004.03.003
















http://link.springer.com/10.1007/s00109-012-0863-6

https://doi.org/10.1007/s00109-012-0863-6


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