Assessing phage-host population dynamics by reintroducing virulent viruses to synthetic microbiomes.

Microbiomes feature complex interactions between diverse bacteria and bacteriophages. Synthetic microbiomes offer a powerful way to study these interactions; however, a major challenge is obtaining a representative bacteriophage population during the bacterial isolation process. We demonstrate that colony isolation reliably excludes virulent viruses from sample sources with low virion-to-bacteria ratios such as feces, creating "virulent virus-free" controls. When the virulent dsDNA virome is reintroduced to a 73-strain synthetic gut microbiome in a bioreactor model of the human colon, virulent viruses target susceptible strains without significantly altering community structure or metabolism. In addition, we detected signals of prophage induction that associate with virulent predation. Overall, our findings indicate that dilution-based isolation methods generate synthetic gut microbiomes that are heavily depleted, if not devoid, of virulent viruses and that such viruses, if reintroduced, have a targeted effect on community assembly, metabolism, and prophage replication.

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis.

By integrating continuous cell cultures with continuous purification methods, process yields and product quality attributes have been improved over the last 10 years for recombinant protein production. However, for the production of viral vectors such as Modified Vaccinia virus Ankara (MVA), no such studies have been reported although there is an increasing need to meet the requirements for a rising number of clinical trials against infectious or neoplastic diseases. Here, we present for the first time a scalable suspension cell (AGE1.CR.pIX cells) culture-based perfusion process in bioreactors integrating continuous virus harvesting through an acoustic settler with semi-continuous chromatographic purification. This allowed obtaining purified MVA particles with a space-time yield more than 600% higher for the integrated perfusion process (1.05 × 1011 TCID50 /Lbioreactor /day) compared to the integrated batch process. Without further optimization, purification by membrane-based steric exclusion chromatography resulted in an overall product recovery of 50.5%. To decrease the level of host cell DNA before chromatography, a novel inline continuous DNA digestion step was integrated into the process train. A detailed cost analysis comparing integrated production in batch versus production in perfusion mode showed that the cost per dose for MVA was reduced by nearly one-third using this intensified small-scale process.

Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy.

As gene delivery tools, lentiviral vectors (LV) have broad applications in chimeric antigen receptor therapy (CAR-T). Large-scale production of functional LV is limited by the adherent, serum-dependent nature of HEK293T cells used in the manufacturing. HEK293T adherent cells were adapted to suspension cells in a serum-free medium to establish large-scale processes for functional LV production in a stirred bioreactor without micro-carriers. The results showed that 293 T suspension was successfully cultivated in F media (293 CD05 medium and SMM293-TII with 1:1 volume ratio), and the cells retained the capacity for LV production. After cultivation in a 5.5 L bioreactor for 4 days, the cells produced 1.5 ± 0.3 × 107 TU/mL raw LV, and the lentiviral transduction efficiency was 48.6 ± 2.8% in T Cells. The yield of LV equaled to the previous shake flask. The critical process steps were completed to enable a large-scale LV production process. Besides, a cryopreservation solution was developed to reduce protein involvement, avoid cell grafting and reduce process cost. The process is cost-effective and easy to scale up production, which is expected to be highly competitive.

Bioreactor production of rVSV-based vectors in Vero cell suspension cultures.

The Vero cell line is the most used continuous cell line in viral vaccine manufacturing. This adherent cell culture platform requires the use of surfaces to support cell growth, typically roller bottles, or microcarriers. We have recently compared the production of rVSV-ZEBOV on Vero cells between microcarrier and fixed-bed bioreactors. However, suspension cultures are considered superior with regard to process scalability. Therefore, we further explore the Vero suspension system for recombinant vesicular stomatitis virus (rVSV)-vectored vaccine production. Previously, this suspension cell line was only able to be cultivated in a proprietary medium. Here, we expand the adaptation and bioreactor cultivation to a serum-free commercial medium. Following small-scale optimization and screening studies, we demonstrate bioreactor productions of highly relevant vaccines and vaccine candidates against Ebola virus disease, HIV, and coronavirus disease 2019 in the Vero suspension system. rVSV-ZEBOV, rVSV-HIV, and rVSVInd -msp-SF -Gtc can replicate to high titers in the bioreactor, reaching 3.87 × 107 TCID50 /ml, 2.12 × 107 TCID50 /ml, and 3.59 × 109 TCID50 /ml, respectively. Furthermore, we compare cell-specific productivities, and the quality of the produced viruses by determining the ratio of total viral particles to infectious viral particles.

Antiviral application of colloidal and immobilized silver nanoparticles.

This study explored the application of colloidal and immobilized silver nanoparticles (AgNPs) for inactivation of bacteriophages. Coliphages that are commonly used as indicators for enteric viruses, were used in this study. Colloidal AgNPs were synthesized via a chemical reduction approach using sodium borohydride as reducing agent and trisodium citrate as stabilizing agent. AgNP-immobilized glass substrate was prepared by immobilizing AgNPs on amine-functionalized glass substrate by post-immobilization method. The AgNP-immobilized glass substrate was also tested so as to minimize the release of AgNPs in the treated water. The characterization of AgNPs and the AgNP-immobilized glass surface was done using field emission gun-transmission electron microscopy and scanning electron microscopy. Studies conducted with varying concentrations of colloidal AgNPs displayed good antiviral activity for MS2 and T4 bacteriophage. Colloidal AgNPs at a dose of 60 µg ml-1 could completely inactivate MS2 and T4 bacteriophage within 30 and 50 min with an initial concentration of 103 PFU ml-1. Contaminated water (100 ml) in an unstirred batch reactor with an initial bacteriophage concentration of 103 PFU ml-1 could be inactivated by the AgNP-immobilized glass substrate (1 cm × 1 cm, containing 3.7 µg cm-2 silver) suspended centrally in the batch reactor. Complete 3-Log bacteriophage inactivation was achieved within 70 and 80 min for MS2 and T4 bacteriophage, respectively, while the aqueous silver concentration was less than 25 µg l-1. This is significantly lower than the recommended standard for silver in drinking water (i.e. 100 µg l-1, US EPA). Thus, AgNP-immobilized glass may have good potential for generating virus-free drinking water.

A CFD model for predicting protein aggregation in low-pH virial inactivation for mAb production.

Significant amounts of soluble product aggregates were observed in the low-pH viral inactivation (VI) operation during an initial scale-up run for an immunoglobulin-G 4 (IgG4) monoclonal antibody (mAb IgG4-N1). Being earlier in development, a scale-down model did not exist, nor was it practical to use costly Protein A eluate (PAE) for testing the VI process at scale, thus, a computational fluid dynamics (CFD)-based high-molecular weight (HMW) prediction model was developed for troubleshooting and risk mitigation. It was previously reported that the IgG4-N1 molecules upon exposure to low pH tend to change into transient and partially unfolded monomers during VI acidification (i.e., VIA) and form aggregates after neutralization (i.e., VIN). Therefore, the CFD model reported here focuses on the VIA step. The model mimics the continuous addition of acid to PAE and tracks acid distribution during VIA. Based on the simulated low-pH zone (≤pH 3.3) profiles and PAE properties, the integrated low-pH zone (ILPZ) value was obtained to predict HMW level at the VI step. The simulations were performed to examine the operating parameters, such as agitation speed, acid addition rate, and protein concentration of PAE, of the pilot scale (50-200 L) runs. The conditions with predictions of no product aggregation risk were recommended to the real scale-up runs, resulted in 100% success rate of the consecutive 12 pilot-scale runs. This study demonstrated that the CFD-based HMW prediction model could be used as a tool to facilitate the scale up of the low-pH VI process directly from bench to pilot/production scale.

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane.

The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.

Performance of an acoustic settler versus a hollow fiber-based ATF technology for influenza virus production in perfusion.

Process intensification and integration is crucial regarding an ever increasing pressure on manufacturing costs and capacities in biologics manufacturing. For virus production in perfusion mode, membrane-based alternating tangential flow filtration (ATF) and acoustic settler are the commonly described cell retention technologies. While acoustic settlers allow for continuous influenza virus harvesting, the use of commercially available membranes for ATF systems typically results in the accumulation of virus particles in the bioreactor vessel. Accordingly, with one single harvest at the end of a cultivation, this increases the risk of lowering the product quality. To assess which cell retention device would be most suitable for influenza A virus production, we compared various key performance figures using AGE1.CR.pIX cells at concentrations between 25 and 50 × 106 cells/mL at similar infection conditions using either an ATF system or an acoustic settler. Production yields, process-related impurities, and aggregation of viruses and other large molecules were evaluated. Taking into account the total number of virions from both the bioreactor and the harvest vessel, a 1.5-3.0-fold higher volumetric virus yield was obtained for the acoustic settler. In addition, fewer large-sized aggregates (virus particles and other molecules) were observed in the harvest taken directly from the bioreactor. In contrast, similar levels of process-related impurities (host cell dsDNA, total protein) were obtained in the harvest for both retention systems. Overall, a clear advantage was observed for continuous virus harvesting after the acoustic settler operation mode was optimized. This development may also allow direct integration of subsequent downstream processing steps. KEY POINTS: • High suspension cell density, immortalized avian cell line, influenza vaccine.

Evaluation of screening platforms for virus-like particle production with the baculovirus expression vector system in insect cells.

Recombinant protein and virus-like particle (VLP) production based on the baculovirus expression vector system is fast, flexible, and offers high yields. Independent from the product, a multitude of parameters are screened during process development/optimisation. Early development acceleration is a key requirement for economic efficiency, and µ-scale bioreactor systems represent an attractive solution for high-throughput (HTP) experimentation. However, limited practical knowledge is available on the relevance and transferability of screening data to pilot scales and manufacturing. The main goal of the present study was to evaluate a HTP µ-bioreactor platform with respect to its aptitude as a screening platform mainly based on transferability of results to benchtop bioreactors representing the conventional production regime. Second question was to investigate to what extent the online sensors of the µ-bioreactor contribute to process understanding and development. We demonstrated that transferability of infection screening results from the HTP µ-bioreactor scale to the benchtop bioreactor was equal or better than that from shaker cultivation. However, both experimental setups turned out to be sub-optimal solutions that only allowed for a first and rough ranking with low relevance in the case of absolute numbers. Bioreactor yields were up to one order of magnitude higher than the results of screening experiments.

Environmental algal phage isolates and their impact on production potential for food and biofuel applications.

AIMS: The United States Department of Energy is aiming to bring microalgal biofuels into commercial use by 2030 at the price of $3 per gasoline gallon equivalent. Large-scale production of biofuel faces many challenges including naturally occurring algal phages; and characterizing this threat is the aim of this study. METHODS AND RESULTS: Bench-scale experiments were performed to study the impact of viral infectivity on the production of microalgal in bioreactors. All environmental samples were tested positive for algal phages which showed various levels of infectivity against Synechocystis PCC 6803 and the environmental isolates of microalgae. The viral attachment to algal cells was observed under transmission electron microscopy (TEM) and to determine the shape and size of the viral particles. All the viruses detected were c. 50-60 nm icosahedral particles. Viral infection resulted in 48% reduction in the biomass of the infected algal culture in 22 days. CONCLUSIONS: This study has lead to the conclusion that the microalgal phages prevalent in natural environment may cause infections in broad range of microalgae used for biofuel production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has detected and quantified the phages that can infect algal populations in natural freshwater habitats and laboratory cultures of microalgal strains. The impact of viral threat to health of commercial algal production operations has been identified in this study.

A narrow residence time incubation reactor for continuous virus inactivation based on packed beds.

A narrow residence time distribution (RTD) is highly desirable for continuous processes where a strict incubation time must be ensured, such as continuous virus inactivation. A narrow RTD also results in faster startup and shut down phases and limits the broadening of potential disturbances in continuous processes. A packed bed reactor with non-porous inert beads was developed to achieve narrow RTDs. The performance was defined as the ratio between the onset of the cumulative RTD and the median residence time (tx%/t50%). Laboratory-scale packed columns were used to study the influence of the column parameters on the RTD. A larger column with a void volume of 0.65 L and a length of 89 cm, packed with beads in a size range of 125 to 250 µm, achieved t0.5%/t50% >0.93 across flow rates from 0.1 to 9.8 mL/min. The RTD was significantly narrower than the RTDs of other reactor designs, such as the Coiled Flow Inverter and Jig in a Box. The pressure drop remained under 3 kPa for all tested flow rates. Fluorescent nanoparticles (30 and 200 nm) were used to mimic viruses. These two sizes showed less than 2% difference in terms of t1%/t50% and t0.01%/t50% scores. These results indicated that viruses travelled through the column at rates independent of size. This proposal of packed beds as incubation chambers for continuous virus inactivation is simple, scalable, and can be realized as single-use devices. Due to the low pressure drop, the system can be easily integrated into a fully continuous process.

Quality by Design-Driven Process Development of Severe Fever With Thrombocytopenia Syndrome Vaccine.

Owing to the biological activity of the vaccine, the complicated production process, sterility, and uniformity of the product, the producing process of the vaccine is complicated and the product quality hard to control. In recent years, with the development of basic science such as cell biology, molecular biology, and metabolic engineering, bioprocess engineering research has developed rapidly. Therefore, U.S. Food and Drug Administration and European Medicines Agency conduct stringent control over the development of biomedical process engineering and product quality. This case study describes an example of Quality by Design-driven process development for manufacturing a human vaccine produced with Vero cells. Cell density in harvest fermentation broth and antigenic titer were chosen as 2 critical quality attributes. The study through 3 rounds design of experiment revealed that H2O2 and cell boost 4 had a significant effect on antigenic titer. Ethanolamine had significant improvement in the final concentration of cells. Through the Monte Carlo simulation, the design spaces and control space of process parameters were determined. A successful validation in a bioreactor was executed to verify the results of a spinner flask. Our investigation presents a successful case of Quality by Design principle, which encourages other researchers to combine the methodology into other biopharmaceutical manufacturing process.

A scale-down model of 4000-L cell culture process for inactivated foot-and-mouth disease vaccine production.

The anticipated increasing demand for inactivated foot-and-mouth (FMD) disease vaccine calls for its larger production capacity, while development of a large-scale process typically requires high running cost and has very limited experimental throughput at manufacturing scale. Thus, an economic scale-down model of representing a large-scale process becomes necessary and essential. In this study, we used a systematic approach to establish a scale-down model representing a 4000-L culture process for FMD vaccine production by suspension BHK-21 cells. In detail, we firstly compared hydrodynamic properties of three bioreactors (14-L, 800-L and 4000-L) under three different conditions (equivalent mixing time, equivalent shear stress and equivalent volumetric power). We figured out equivalent volumetric power (P/V) potentially as an appropriate scale-down strategy, since it resulted in comparable calculated hydrodynamic parameters among three bioreactors. Next, we used computational fluid dynamics (CFD) simulation to provide more details about hydrodynamic environments inside the bioreactors, which supports the reliability of this scale-down strategy. Finally, we compared cell growth, metabolites, vaccine productivity and product quality attributes during FMD vaccine production by BHK-21 cells and observed very close performances among three bioreactors, which once again demonstrates the robustness of this scale-down model. This scale-down strategy can be applied to study variations and critical quality attributes (CQAs) in the resultant production process based on quality by design (QbD) principles, aiming at further more efficient optimization of vaccine production.

Viral composition and context in metagenomes from biofilm and suspended growth municipal wastewater treatment plants.

Wastewater treatment plants (WWTPs) contain high density and diversity of viruses which can significantly impact microbial communities in aquatic systems. While previous studies have investigated viruses in WWTP samples that have been specifically concentrated for viruses and filtered to exclude bacteria, little is known about viral communities associated with bacterial communities throughout wastewater treatment systems. Additionally, differences in viral composition between attached and suspended growth wastewater treatment bioprocesses are not well characterized. Here, shotgun metagenomics was used to analyse wastewater and biomass from transects through two full-scale WWTPs for viral composition and associations with bacterial hosts. One WWTP used a suspended growth activated sludge bioreactor and the other used a biofilm reactor (trickling filter). Myoviridae, Podoviridae and Siphoviridae were the dominant viral families throughout both WWTPs, which are all from the order Caudovirales. Beta diversity analysis of viral sequences showed that samples clustered significantly both by plant and by specific sampling location. For each WWTP, the overall bacterial community structure was significantly different than community structure of bacterial taxa associated with viral sequences. These findings highlight viral community composition in transects through different WWTPs and provide context for dsDNA viral sequences in bacterial communities from these systems.

Purification of rabies virus produced in Vero cells grown in serum free medium.

Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Vaccination against rabies is still the sole efficient way to fight against the disease. Cell culture vaccines are recommended by World Health Organization (WHO) for pre and post exposure prophylaxis; among them Vero cell rabies vaccines which are used worldwide. In this work we studied the purification of inactivated rabies virus produced in Vero cells grown in animal component free conditions, using different methods. Cells were grown in VP-SFM medium in stirred bioreactor, then infected at an MOI of 0.05 with the LP2061 rabies virus strain. Collected harvests were purified by zonal centrifugation, and by chromatography supports, namely the Capto Core 700 and the monolithic CIM-QA column. Generated data were compared in terms of residual DNA level, host cell proteins (HCP) level and the overall recovery yield. Rabies virus purification using the monolithic column resulted in the highest antigen recovery yield, equal to 94%. Capto Core 700 showed a lower yield, about 84%; whereas the purification yield by zonal centrifugation was equal to 60%. In terms of host cell residual DNA removal, zonal centrifugation was the most efficient method; the removal yield was equal to 88.5%; elimination of host cell DNA was slightly lower when using the monolithic CIM-QA (equal to 73%). Whereas Capto Core 700 showed the lowest level (49.2%). Host cell protein removal varied between 92.6% for the monolithic column and 78.6% for the zonal centrifugation. Capto Core 700 eliminated 86.5% of HCP.

Accelerating bioprocess development by analysis of all available data: A USP case study.

Bioprocess development generates extensive datasets from different unit operations and sources (e.g. time series, quality measurements). The development of such processes can be accelerated by evaluating all data generated during the experimental design. This can only be achieved by having a clearly defined data logging and analysis strategy. The latter is described in this manuscript. It consists in a combination of a feature based approach along with principal component analysis and partial least square regression. Application of this combined strategy is illustrated by applying it in an upstream processing (USP) case study. Data from the development and optimization of an animal component free USP of Sabin inactivated poliovirus vaccine (sIPV) was evaluated. During process development, 26 bioreactor runs at scales ranging from 2.3 to 16 L were performed. Several operational parameters were varied, and data was routinely analyzed following a design of experiments (DoE) methodology. With the strategy described here, it became possible to scrutinize all data from the 26 runs in a single data study. This included the DoE response parameters, all data generated by the bioreactor control systems, all offline data, and its derived calculations. This resulted in a more detailed, reliable and exact view on the most important parameters affecting bioreactor performance. In this case study, the strategy was applied for the analysis of previously produced data. Further development will use this data analysis methodology for continuous enhancing and accelerating process development, intensified DoE and integrated process modelling.

Influenza A virus production in a single-use orbital shaken bioreactor with ATF or TFF perfusion systems.

Driven by the concept of plug-and-play cell culture-based viral vaccine production using disposable bioreactors, we evaluated an orbital shaken bioreactor (OSB) for human influenza A virus production at high cell concentration. Therefore, the OSB model SB10-X was coupled to two hollow fiber-based perfusion systems, namely, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). The AGE1.CR.pIX avian suspension cells grew to 50 × 106 cells/mL in chemically defined medium, maintaining high cell viabilities with an average specific growth rate of 0.020 h-1 (doubling time = 32 h). Maximum virus titers in the range of 3.28-3.73 log10(HA units/100 µL) were achieved, corresponding to cell-specific virus yields of 1000-3500 virions/cell and productivities of 0.5-2.2 × 1012 virions/L/d. This clearly demonstrates the potential of OSB operation in perfusion mode, as results achieved in a reference OSB batch cultivation were 2.64 log10(HA units/100 µL), 1286 virions/cell and 1.4 × 1012 virions/L/d, respectively. In summary, the SB10-X bioreactor can be operated with ATF and TFF systems, which is to our knowledge the first report regarding OSB operation in perfusion mode. Moreover, the results showed that the system is a promising cultivation system for influenza A virus vaccine production. The OSB disposable bioreactor has the potential for simplifying the scale-up from shake flasks to the large-scale bioreactor, facilitating rapid responses in the event of epidemics or pandemics.

Development of suspension adapted Vero cell culture process technology for production of viral vaccines.

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40-44 h. Much higher cell density (8 × 106 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 106 cells/mL.

Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media.

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective. The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated. In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 106 cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers. Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ±â€¯0.5 × 105 cells/mL resulted in a virus titer higher than 107 FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ±â€¯0.5 × 107 FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca++ and Mg++. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.

Semi-perfusion cultures of suspension MDCK cells enable high cell concentrations and efficient influenza A virus production.

Control and prevention of rapid influenza spread among humans depend on the availability of efficient and safe seasonal and pandemic vaccines, made primarily from inactivated influenza virus particles. Current influenza virus production processes rely heavily on embryonated chicken eggs or on cell culture as substrate for virus propagation. Today's efforts towards process intensification in animal cell culture could innovate viral vaccine manufacturing using high-yield suspension cells in high cell density perfusion processes. In this work, we present a MDCK cell line adapted to grow as single cell suspension with a doubling time of less than 20 h, achieving cell concentrations over 1 × 107 cells/mL in batch mode. Influenza A virus titer obtained in batch infections were 3.6 log10(HAU/100 µL) for total- and 109 virions/mL for infectious virus particles (TCID50), respectively. In semi-perfusion mode concentrations up to 6 × 107 cells/mL, accumulated virus titer of 4.5 log10(HAU/100 µL) and infectious titer of almost 1010 virions/mL (TCID50) were possible. This exceeds results reported previously for cell culture-based influenza virus propagation by far and suggests perfusion cultures as the preferred method in viral vaccine manufacturing.