Suspension-Vero cell cultures as a platform for viral vaccine production

Since Vero cells are currently considered as an acceptable cell substrate to produce a wide range of viruses, we developed a virus production platform using Vero cells adapted to grow in suspension in serum-free media. After adapting anchorage-dependent Vero cells to grow as a free-cell suspension, vesicular stomatitis virus, herpes simplex virus 1 and polio virus 1 production rates were evaluated in batch cultures using spinner flasks and perfused cultures in a bioreactor. The achieved results constitute valuable information for the development of a low-cost high-productivity process using a suspension culture of Vero cells to produce viral vaccines.     

Statistical optimization of influenza H1N1 production from batch cultures of suspension Vero cells (sVero)

Efficient vaccine production requires the growth of large quantities of virus produced with high yield from a safe host system. Human influenza vaccines are produced in embryonated chicken eggs. However, over the last decade many efforts have allowed the establishment of cell culture-derived vaccines.We generated a Vero cell line adapted to grow in suspension (sVero) in a serum-free medium and evaluated it for its potential as host cell for influenza vaccine production. Initially we studied the capacity of sVero cells to grow in the presence of incremental concentrations of trypsin. In comparison with adherent Vero cells (aVero), we found that sVero cells maintain their growth kinetics even with a three-fold increase in trypsin concentration.The influence of the conditions of infection on the yield of H1N1 produced in serum-free suspension cultures of sVero cells was investigated by a 2² full factorial experiment with center point. Each experiment tested the influence of the multiplicity of infection (m.o.i.) and trypsin concentration, on production yields at two levels, in four possible combinations of levels and conditions, plus a further combination in which each condition was set in the middle of its extreme levels.On the basis of software analysis, a combination of m.o.i. of 0.0066 TCID_50%/cell and trypsin concentration of 5 μg/1.0 × 10⁶ cells with a desirability of 0.737 was selected as the optimized condition for H1N1 production in sVero cells.Our results show the importance of proper selection of infection conditions for H1N1 production on sVero cells in serum-free medium.     

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.     

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 ± 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 ± 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.     

Efficient influenza A virus production in high cell density using the novel porcine suspension cell line PBG.PK2.1

Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza virus production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine production and efficiency.Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for virus production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza virus production was improved after virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log₁₀(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 10⁶ cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log₁₀(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza virus vaccine production.     

An animal component free medium that promotes the growth of various animal cell lines for the production of viral vaccines

IPT-AFM is a proprietary animal component free medium that was developed for rabies virus (strain LP 2061) production in Vero cells. In the present work, we demonstrated the versatility of this medium and its ability to sustain the growth of other cell lines and different virus strains. Here, three models were presented: Vero cells/rabies virus (strain LP 2061), MRC-5 cells/measles virus (strain AIK-C) and BHK-21 cells/rabies virus (strain PV-BHK21). The cell lines were first adapted to grow in IPT-AFM, by progressive reduction of the amount of serum in the culture medium. After their adaptation, BHK-21 cells grew in suspension by forming clumps, whereas MRC-5 cells remained adherent. Then, kinetics of cell growth were studied in agitated cultures for both cell lines. In addition, kinetics of virus replication were investigated.     

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

The increasing importance of viral vaccine manufacturing has driven the need for high cell density process optimization that allows for higher production levels. Vero cells are one of the more popular adherent cell lines used for viral vaccine production. However, production is limited due to the logistical limitations surrounding adherent cell line processes, such as large equipment footprints, time and labor-intensive processes, and larger costs per dose. We have addressed this limitation with the establishment of a viral vaccine production system utilizing the novel single use scale-X™ 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 present the results from a campaign featuring a proprietary Vero cell line for production of a live recombinant 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 bioreactor were performed, and on average, the single use bioreactor produced 2–4 logs higher titers per surface 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.     

Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line

Cell culture-based manufacturing of influenza vaccines is ideally based on easily scalable platforms using suspension cells that grow in chemically defined media. Consequently, different adherent cell lines selected for high virus yields were adapted to grow in suspension culture. This includes the MDCK suspension cell line MDCK.SUS2, which was shown to be a suitable substrate for influenza virus propagation in previous studies. In this study, we investigated options for further improvement of influenza A/PR/8 (H1N1) virus titres and cell-specific virus yields. Best results were achieved by performing a 1:2 dilution with fresh medium at time of infection. In shake flask cultivations, even for multiplicities of infection as low as 10⁻⁵, all cells were infected at 36 h post infection as determined by flow cytometry. In addition, these cells showed a better viability than cells infected without previous washing steps, which was reflected by a reduced level of apoptotic cells, and virus yields exceeding 3 log₁₀ HAU/100 μL. Comparison of bioreactor infections of MDCK.SUS2 cells to the parental adherent MDCK cells showed similar HA titres of 2.94 and 3.15 log₁₀ HAU/100 μL and TCID₅₀ of 1 × 10⁹ and 2.37 × 10⁹ infectious virions/mL. Surprisingly, virus-induced apoptosis differed between the two cell lines, with the MDCK.SUS2 cells showing a much stronger apoptosis induction than the adherent MDCK cells. Obviously, despite their resistance to anoikis, the suspension cells were more susceptible to virus-induced apoptosis. Whether this is related to the adaptation process itself and/or to changes in cell survival pathways influenced by adhesion molecules or influenza virus proteins needs to be clarified in additional studies.     

Scalable culture systems using different cell lines for the production of Peste des Petits ruminants vaccine

Peste des Petits ruminants (PPR) is considered as one of the major constraints to the productivity of small ruminants in Africa and Asian countries. Currently PPR control is done by vaccination with an attenuated PPR strain (Nigeria 75/1) produced in monolayers of Vero cells grown in roller bottles or static flasks. This work focuses on the production of a PPR vaccine strain using stirred conditions as an advanced option for process scale-up. Non-porous microcarriers (Cytodex-1) were used to support Vero cell growth in suspension cultures. The use of Ex-Cell medium could improve cell specific productivities obtained with standard serum containing medium, independently of the type of system used, i.e. static as well as suspension stirred cultures. As an alternative, several cell lines adapted to grow as single cells in suspension (CHO-K1, BHK-21A and 293) and another anchorage-dependent (MRC-5) were evaluated in their capacity to produce a PPR vaccine. BHK-21A and 293 cells grown as single-cell suspension in serum free medium were both suited to produce PPR vaccine with productivities similar to Vero cells, namely 10(6)TCID(50)/mL. However, for the 293 cells, these results were only obtained 2-3 days later. CHO-K1 and MRC-5 cells have shown not to be suitable to adequately produce this virus. These results provide further insights into the feasibility of applying microcarrier cell culture technology to produce PPR vaccine in Vero cells as well as in the alternative use of single-cell suspension cultures of BHK-21A, significantly simplifying the existing production process.     

Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells

The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging.Here we present a production platform based on BHK-21 suspension (BHK-21_SUS) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1–48 PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKV^PE) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21_SUS cells with ZIKV^PE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9 × 10³ PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2 × 10⁷ cells/mL, and virus titers of 3.9 × 10⁷ PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3 PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards process development for manufacturing inactivated or live-attenuated ZIKV vaccines.     

Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing

Cell culture processes offer an attractive alternative to conventional chicken egg-based influenza vaccine production methods. However, most protocols still rely on the use of adherent cells, which makes process scale-up a challenging issue. In this study, it is demonstrated that the HEK-293 human cell line is able to efficiently replicate influenza virus. Production in serum-free suspension of HEK-293 cultures resulted in high titers of infectious influenza viruses for different subtypes and variants including A/H1, A/H3 and B strains. After virus adaptation and optimization of infection conditions, production in 3-L bioreactor resulted in titers of up to 10⁹ IVP/mL demonstrating the scale-up potential of the process.     

Long-term stability of Vero cell-derived inactivated Japanese encephalitis vaccine prepared using serum-free medium

We established a method of producing a Vero cell-derived Japanese encephalitis vaccine using serum-free medium, and tested its stability using various stabilizers during the inactivation process and storage at 4 degrees C and 28 degrees C. Similar to previously reported results of cell culture in serum-containing medium, Vero cells were cultured in a serum-free medium multiplied well, and the viral yield was successfully increased to about 10(9)PFU/ml. Following formalin-inactivation and purification via ethanol precipitation and sucrose density ultracentrifugation of the virus solution, the vaccine had the same quality as, and higher immunogenicity, the mouse brain-derived vaccine in current use. Testing of several stabilizers showed that the addition of 0.5% glycine during the virus inactivation process facilitated the maintenance of immunogenicity for a long period of time. Furthermore, the addition of 0.5% glycine and 1.0% sorbitol as vaccine stabilizers after purification led to the maintenance of immunogenicity for 1 year, not dependent on the storage temperature (4 degrees C or 28 degrees C). These results indicate that, in contrast to the current mouse brain-derived vaccine, the Vero cell-derived vaccine can be prepared using serum-free medium containing no animal-derived components, and that the vaccine can be stored at room temperature by adding stabilizers, suggesting the possibility of producing room temperature-stable vaccines.     

Production of high titer attenuated poliovirus strains on the serum-free PER.C6® cell culture platform for the generation of safe and affordable next generation IPV

BackgroundAs poliovirus eradication draws closer, alternative Inactivated Poliovirus Vaccines (IPV) are needed to overcome the risks associated with continued use of the Oral Poliovirus Vaccine and of neurovirulent strains used during manufacture of conventional (c) IPV. We have previously demonstrated the susceptibility of the PER.C6^® cell line to cIPV strains; here we investigated the suspension cell culture platform for growth of attenuated poliovirus strains.MethodsWe examined attenuated Sabin strain productivity on the PER.C6^® cell platform compared to the conventional Vero cell platform. The suitability of the suspension cell platform for propagation of rationally-attenuated poliovirus strains (stabilized Sabin type 3 S19 derivatives and genetically attenuated and stabilized MonoCre(X) strains), was also assessed. Yields were quantified by infectious titer determination and D-antigen ELISA using either serotype-specific polyclonal rabbit sera for Sabin strains or monoclonal cIPV-strain-specific antibodies for cIPV, S19 and MonoCre(X) strains.ResultsPER.C6^® cells supported the replication of Sabin strains to yields of infectious titers that were in the range of cIPV strains at 32.5 °C. Sabin strains achieved 30-fold higher yields (p < 0.0001) on the PER.C6^® cell platform as compared to the Vero cell platform in infectious titer and D-antigen content. Furthermore, Sabin strain productivity on the PER.C6^® cell platform was maintained at 10 l scale. Yields of infectious titers of S19 and MonoCre(X) strains were 0.5–1 log₁₀ lower than seen for cIPV strains, whereas D-antigen yield and productivities in doses/ml using rationally-attenuated strains were in line with yields reported for cIPV strains.ConclusionsSabin and rationally-attenuated polioviruses can be grown to high infectious titers and D-antigen yields. Sabin strain infection shows increased productivity on the PER.C6^® cell platform as compared to the conventional Vero cell platform. Novel cell platforms with the potential for higher yields could contribute to increased affordability of a next generation of IPV vaccines needed for achieving and maintaining poliovirus eradication.     

A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions

Rabies virus strain production in Vero cells grown on Cytodex 1 in a 2 L stirred tank bioreactor and in a medium free of components of human or animal origin (VP-SFM) is described. Cell banking procedure in VP-SFM supplemented with an animal components free mixture (10%DMSO+0.1%methylcellulose) was reported and cell growth after revitalization was assessed. Vero cells exhibited growth performances (specific growth rate and cell division number) similar to that obtained in serum containing medium. To design a scalable process that is totally free of animal-derived substances, two proteases: TrypLE Select and Accutase, were assessed as an alternative to trypsin which is routinely used for cell passage. Growth performance of Vero cells grown in VP-SFM and MEM+10% fetal calf serum (FCS) over four passages and subcultivated with either TrypLE Select or Accutase was evaluated. TrypLE Select showed the best performance in terms of specific growth rate and cell division number. Kinetics of cell growth and rabies virus production (LP2061/Vero strain) were investigated in spinner flask and in a 2 L bioreactor. In spinner flask, a maximal cell density level of 1.85x10(6) cells/mL was achieved when the cells were grown in VP-SFM on 2 g/L Cytodex 1. Cell infection experiments conducted at an MOI of 0.3 and without the medium exchange step, typically needed for serum containing rabies virus production, resulted in a maximal virus titer equal to 2x10(7) (Fluorescent Focus Unit) FFU/mL. In stirred tank bioreactor, Vero cell growth in VP-SFM on 3 g/L Cytodex 1 was shown to be sensitive to the aeration mode. Sparging the culture was detrimental for cell growth, whereas cell density level was greatly enhanced when only headspace aeration was used. A cell density level of 2.6x10(6) cells/mL was obtained when the cells were grown on 3g/L Cytodex 1 and in batch culture mode. Cell infection at an MOI of 0.1 without any medium exchange, yielded a maximal rabies virus titer of 2.4x10(7) FFU/mL. Furthermore, Vero cell growth in a 2 L bioreactor using recirculation culture mode during cell proliferation step and perfusion for virus multiplication phase was investigated. In comparison to batch culture, a higher cell density level that was equal to 5x10(6) cells/mL was reached. Cell infection under conditions similar to batch culture, resulted in a maximal virus titer equal to 1.38x10(8) FFU/mL. The potency of the pooled inactivated virus harvests showed an activity of 2.58 IU/mL which was comparable to that obtained in serum supplemented medium.     

Process intensification for Peste des Petites Ruminants Virus vaccine production

Process intensification for Peste des Petites Ruminants Virus (PPRV) vaccine production in anchorage dependent Vero cells is challenging, involving substantial amount of bioprocess development.In this study, we describe the implementation of a new, scalable bioprocess for PPRV vaccine production in Vero cells using serum-free medium (SFM), microcarrier technology in stirred-tank bioreactors (STB), in-situ cell detachment from microcarriers and perfusion. Vero cells were successfully adapted to ProVero™-1 SFM, reaching growth rates similar to serum-containing cultures (0.030 1/h vs 0.026 1/h, respectively). An in-situ cell detachment method was successfully implemented, with efficiencies above 85%. Up to 2.5-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining perfusion with the in-situ cell detachment method enabled the scale-up to 20 L STB directly from a 2 L STB, surpassing the need for a mid-scale platform (i.e. 5 L STB) and thus reducing seed train duration. Head-to-head comparison of cell growth and PPRV production in the 2 L and 20 L STB was performed, and no significant differences could be observed. Estimated infectious PPRV titers in Tissue Culture Infection Dose (TCID₅₀) (TCID₅₀/mL = 5 × 10⁶ and TCID₅₀/cell = 5) are within the log-range reported in literature for PPRV production in STB and SFM by Silva et al. (2008), thus confirming the feasibility and scalability of the seed train designed [1].The novel and scalable vaccine production process herein proposed has the potential to assist the upcoming Peste des Petites Ruminants (PPR) Global Eradication Program (targeted by FAAO for 2030) by providing African local and/or regional manufacturers with a platform capable of generating over 25,000 doses of Nigeria 75/1 strain in just 19 days using a 20 L STB.     

Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development

Enterovirus 71 (EV71) is an enterovirus that could lead to severe neurological disorders and fatalities. The inactivated vaccine is an appropriate EV71 vaccine format for meeting current needs. Large-scale preparation of the inactivated EV71vaccine depends on a scalable cell culture system for industrial mass production. In this paper, Vero cells were found to produce higher titers of EV71 than did MRC-5 and WI-38 cells. High-density microcarrier Vero cell cultures were established using 5g/L Cytodex 1 microcarriers and found to promote the release of EV71s from infected Vero cells. For the large-scale production of the inactivated vaccine antigen, the extracellular virus titers produced in the 2L bioreactor were found to be 10 times lower than the spinner flask culture but improved by 30-folds using glucose/glutamine feedings during infection. A serum-free Vero cell microcarrier culture was also established in the bioreactor, yielding a high-titer of 5.8 x 10(7) TCID50/mL for EV71 production. The immunogenicity of the inactivated virions produced in serum-free culture elicited a slightly higher level of neutralizing antibody response in immunized mice. These results constitute valuable information on the development of a large-scale microcarrier cell culture process for producing inactivated EV71 vaccine.     

Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a non-animal, non-human and non-plant-derived medium

For the production of a chemically inactivated Parapoxvirus ovis (PPVO), an adherent bovine kidney cell line was cultivated on Cytodex-3 microcarriers in suspension culture. The inactivated and purified virus particles have shown immune modulatory activity in several animal models. PPVO was produced by a biphasic batch process at the 3.5 and 10 L scale. Aeration was realised by bubble-free membrane oxygenation via a tube stator with a central two-blade anchor impeller. In order to increase efficiency, process robustness and safety, the established process was optimised. The cell line was adapted to a protein-free medium (except recombinant insulin) in order to increase biosafety. A scale up to a 50 L pilot plant with direct cell expansion was performed successfully. In parallel, the biphasic batch process was optimised with special emphasis on different operating conditions (cell number, Multiplicity of Infection (MOI), etc.) and process management (fed-batch, dialysis, etc.). The quality and concentration of the purified virus particles was assessed by quantitative electron microscopy, residual host cell protein and DNA-content and, finally, biologic activity in a transgenic mouse model. This integrated approach led to a new, safe, robust and highly productive large-scale production process, called "Volume-Expanded-Fed" Batch with cell densities up to 6-7e06 cells/mL. By subsequent dilution of infected cells into the next process scale, an increase in total productivity by a factor of 40 (related to an established biphasic batch process) was achieved.     

Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor

Hollow fiber bioreactors (HFBRs) have been widely described as capable of supporting the production of highly concentrated monoclonal antibodies and recombinant proteins. Only recently HFBRs have been proposed as new single-use platforms for production of high-titer influenza A virus. These bioreactors contain multiple hollow fiber capillary tubes that separate the bioreactor in an intra- and an extra-capillary space. Cells are usually cultured in the extra-capillary space and can grow to a very high cell concentration. This work describes the evaluation of the single-use hollow fiber bioreactor PRIMER HF^® (Biovest International Inc., USA) for production of influenza A virus. The process was setup, characterized and optimized by running a total of 15 cultivations. The HFBRs were seeded with either adherent or suspension MDCK cells, and infected with influenza virus A/PR/8/34 (H1N1), and the pandemic strain A/Mexico/4108/2009 (H1N1). High HA titers and TCID₅₀ of up to 3.87 log₁₀ (HA units/100 μL) and 1.8 × 10¹⁰ virions/mL, respectively, were obtained for A/PR/8/34 influenza strain. Influenza virus was collected by performing multiple harvests of the extra-capillary space during a virus production time of up to 12 days. Cell-specific virus yields between 2,000 and 8,000 virions/cell were estimated for adherent MDCK cells, and between 11,000 and 19,000 virions/cell for suspension MDCK.SUS2 cells. These results do not only coincide with the cell-specific virus yields obtained with cultivations in stirred tank bioreactors and other high cell density systems, but also demonstrate that HFBRs are promising and competitive single-use platforms that can be considered for commercial production of influenza virus.     

Improved poliovirus d-antigen yields by application of different Vero cell cultivation methods

Vero cells were grown adherent to microcarriers (Cytodex 1; 3 g L⁻¹) using animal component free media in stirred-tank type bioreactors. Different strategies for media refreshment, daily media replacement (semi-batch), continuous media replacement (perfusion) and recirculation of media, were compared with batch cultivation. Cell densities increased using a feed strategy from 1 × 10⁶ cells mL⁻¹ during batch cultivation to 1.8, 2.7 and 5.0 × 10⁶ cells mL⁻¹ during semi-batch, perfusion and recirculation, respectively.