Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production

<p>Abstract</p> <p>Background</p> <p>In cell culture-based influenza vaccine production the monitoring of virus titres and cell physiology during infection is of great importance for process characterisation and optimisation. While conventional virus quantification methods give only virus titres in the culture broth, data obtained by fluorescence labelling of intracellular virus proteins provide additional information on infection dynamics. Flow cytometry represents a valuable tool to investigate the influences of cultivation conditions and process variations on virus replication and virus yields.</p> <p>Results</p> <p>In this study, fluorescein-labelled monoclonal antibodies against influenza A virus matrix protein 1 and nucleoprotein were used for monitoring the infection status of adherent Madin-Darby canine kidney cells from bioreactor samples. Monoclonal antibody binding was shown for influenza A virus strains of different subtypes (H1N1, H1N2, H3N8) and host specificity (human, equine, swine). At high multiplicity of infection in a bioreactor, the onset of viral protein accumulation in adherent cells on microcarriers was detected at about 2 to 4 h post infection by flow cytometry. In contrast, a significant increase in titre by hemagglutination assay was detected at the earliest 4 to 6 h post infection.</p> <p>Conclusion</p> <p>It is shown that flow cytometry is a sensitive and robust method for the monitoring of viral infection in fixed cells from bioreactor samples. Therefore, it is a valuable addition to other detection methods of influenza virus infection such as immunotitration and RNA hybridisation. Thousands of individual cells are measured per sample. Thus, the presented method is believed to be quite independent of the concentration of infected cells (multiplicity of infection and total cell concentration) in bioreactors. This allows to perform detailed studies on factors relevant for optimization of virus yields in cell cultures. The method could also be used for process characterisation and investigations concerning reproducibility in vaccine manufacturing.</p

Evaluation of producer cell lines for yellow fever virus production in up to 1 L bioreactor scale

Yellow fever virus (YFV) vaccine is currently produced in embryonated chicken eggs. Following recent outbreaks of flavivirus-related diseases, such as Zika fever, significant efforts are needed towards fast establishment of cell culture-based production processes for attenuated or inactivated virus vaccines. To support the development of such processes, we have screened various cell lines, including adherent and suspension cells, for permissiveness and productivity of YFV. In particular, the parental adherent Vero cell line possesses a reasonable cell-specific productivity of about 13 PFU/cell. However, surface-depended scale-up restricts production processes to roller bottles, microcarrier-based or fixed-bed bioreactors with limited monitoring and excessive efforts for large-scale production. A preferential alternative is the cultivation of single-cells in stirred-tank bioreactors, which can be operated in perfusion mode to achieve higher cell-densities. Towards this process intensification, we have adapted the parental WHO Vero cell line to grow in suspension. However, infection studies of Vero suspension cells with YFV in spinner flasks using chemically defined medium showed a reduced cell-specific titer (2 PFU/cell). Another option might be the use of BHK-21 cells reaching cell-densities above 5 × 106 cells/mL in shake flasks. Infection studies with YFV in small-scale have resulted in a cell-specific productivity of 10 PFU/cell. Thus, infection parameters (time of infection, MOI = ratio of virus to cell) were optimized and subsequently transferred into 1 L bioreactors. Final titer of 5 × 107 PFU/mL could be reached. As a reference, adherent Vero cells were cultivated on Cytodex-1 microcarriers in 1 L scale resulting in a final titer of 2 × 107 PFU/mL. In both cultivations, cell-specific yields were comparable but due to the adjusted MOI of 10-4 in the BHK-21 cultivation, the overall virus production was 50 × higher than for the Vero cultivation on microcarriers. Although BHK-21 cells and their application for human vaccines are controversial with respect to tumorigenicity and oncogenicity, our results show that it may be worth to reconsider this cell line for future production processes

Stirred tanks in cascades and plug-flow tubular bioreactors for continuous production of viral vaccines

Seasonal and emerging viruses are a major threat for human and animal health worldwide. Whole-virus vaccines are currently produced in batch processes that are either egg- or cell culture-based. Therefore, the shift to continuous processing can be a major technological development that can help to reduce the cost of manufacture and increase vaccine accessibility worldwide [1]. Since continuous processes are known to be more efficient than batch at large production volumes, they can be used preferentially for production of highly demanded viral vaccines. One example is the seasonal influenza virus that causes annual epidemics in human populations worldwide and is currently produced in batch mode. Another virus of interest is Modified Vaccinia Ankara (MVA) virus, which can be used for production of recombinant vaccines or viral vectors [2]. Because both are lytic viruses, one approach to produce them is using continuous stirred tank bioreactors (CSTRs) in cascades, where cell propagation and virus replication occurs in separated vessels [3]. Unfortunately, some viruses produce defective interfering particles that then lead to oscillations in virus levels and low overall production yields for the cascade solution [3]. This phenomenon, known as von Magnus effect, can be overcome, if the virus is propagated in a plug-flow tubular bioreactor (PFBR) using a virus stock of defined passage number for the infection. In this work, we describe the establishment of CSTRs in a cascade and a PFBR system for production of MVA and influenza virus, respectively. A semi-continuous two-stage shaken cultivation system (two 100 mL shaker flasks; SSC) was established as screening tool for influenza and MVA virus propagation before scaling to a “real” cascade of CSTRs (two 1 L stirred tank bioreactors). The MVA virus strains MVA-CR19 and MVA-CR19.GFP were used, and propagated in the duck cell line AGE1.CR.pIX (all three from ProBioGen, Berlin). In addition, a PFBR prototype system was constructed for continuous influenza virus production [4]. The system consisted of a 500 mL stirred tank bioreactor (360 mL working volume) connected to a PFBR (211 mL, silicone-based tube, 105 m) and was operated with a nominal flow rate of 12 mL/h. PCR analysis was used to monitor the stability of MVA and influenza viruses. The SSC system resulted in stable production of cells, and influenza virus titers that approached the oscillatory behavior observed in previous experiments [3]. Interestingly, MVA virus cultivated in the SSC system did not show oscillations in the virus titer. Subsequently, production of MVA-CR19 was scaled to the cascade of CSTRs and maintained for 18 days in continuous mode, confirming the absence of a von Magnus effect over 18 days for MVA virus. Also, the PFBR system resulted in stable production of cells, and stable influenza virus titers ranging between 1.5 and 2.5 log10(HA Units/100μL) for pIX and MDCK cells, respectively. Therefore, for the first time, the von Magnus effect of influenza virus observed in a CSTR cascade was overcome using a PFBR. Overall, it was demonstrated that production of MVA and influenza viruses in continuous mode is feasible using either CSTRs in a cascade or a PFBR system, respectively. Both bioreactor systems can be considered as cost-efficient tools for production of viral vaccines in continuous mode. [1] Hill et al. 2016, Curr. Opin. Biotechnol. 42:67-73. [2] Jordan et al. 2013, Viruses 5(1):321–39. [3] Frensing et al. 2013, PLOS ONE 8(9):e72288. [4] Tapia, Genzel, Reichl 2016, Patent Application, PCT/EP2016/060150

Propagation of influenza and MVA virus in cascades of continuous stirred tank bioreactors: challenging the Von Magnus effect

Moving from batch to fully continuously operated upstream processes is one of the big challenges for the coming decades in cell culture-based viral vaccine manufacturing. Continuous processes are known to be more efficient than batch systems for production of large volumes of product, and can therefore be an interesting option for production of highly demanded viral vaccines. One example is the seasonal influenza virus that causes annual epidemics in human populations worldwide and is currently produced in batch processes. Another virus of clinical interest is Modified Vaccinia Ankara (MVA) virus which is a potential platform for recombinant vaccines and can be used as a vector in gene therapy [1]. Continuous propagation of MVA virus seems to be feasible using a new MVA virus strain that can propagate at high yields in non-aggregated avian suspension cells [2]. Because both influenza and MVA are lytic viruses a continuous production strategy was employed that involves cascades of two stirred tank bioreactors, where cell growth and virus propagation occur in separated vessels [3]. However, a possible drawback for continuous virus production is the presence of defective interfering particles among the virus population that cause oscillations in virus levels and low production yields [3], known as Von Magnus effect. In this work, a small scale two-stage cultivation system (two 100 mL shaker flasks; semi-continuous; SSC) was established as screening tool for influenza and MVA virus propagation before scaling to a 1 L continuous two-stage bioreactor system (two 1 L stirred tank bioreactors; TSB). The MVA virus strains MVA-CR19 and MVA-CR19.GFP were used, and propagated 14 days in the duck cell line AGE1.CR.pIX (all three from ProBioGen, Berlin) using the SSC system. Similarly, the influenza virus strain A/PR/8/34 H1N1 (RKI) was propagated 14 days using two different cell lines (MDCK.SUS2 and AGE1.CR.pIX) in the SSC system. From the best screening result, scale-up to the 1 liter TSB was performed with successful virus production in continuous mode for three weeks. PCR analysis was used to monitor the stability of the viruses in continuous culture. The SSC system resulted in stable production of cells, and influenza virus titers that approached the oscillatory behavior observed in previous experiments [3]. Interestingly, MVA virus cultivated in the SSC system did not show oscillations in the virus titer. Additional cultivations of MVA virus in the SSC system showed that different residence times in the virus bioreactor could influence virus titers. Subsequently, production of MVA-CR19 was scaled to the TSB system and maintained for 18 days in continuous mode. MVA virus titers showed 7 days of a transient phase, followed by stable titers that confirmed the absence of a Von Magnus effect over 18 days. A yield comparison between an eight days batch-cycle process and the TSB showed that the space-time yield of the TSB cultivation approached that of two parallel batches at 11 days of virus production. PCR analysis indicated that the reporter gene in MVA-CR19.GFP was maintained stably for the complete cultivation period. Overall, it was demonstrated that production of influenza and MVA viruses in a SSC system is feasible and can be used as a fast and cost-efficient tool for optimizing continuous virus production. Finally, MVA virus is a very promising candidate for production of viral vaccines in cascades of continuous stirred tank bioreactors. [1] Verheust et al. 2012, Vaccine 30(16):2623–32. [2] Jordan et al. 2013, Viruses 5(1):321–39. [3] Frensing et al. 2013, PLOS ONE 8(9):e72288. [ 4] Westgate and Emery 1990, Biotech & Bioeng 35(5):437-53

Dynamics of intracellular metabolite pools in MDCK suspension cells during growth and influenza virus infection

Influenza virus infections are responsible for millions of flu cases with hundred thousands of deaths worldwide [1]. Additionally, pandemic outbreaks of aggressive influenza virus strains are very dangerous both for livestock and human population. Seasonal vaccination campaigns are in place to reduce infections, especially among young, old or immunodeficient individuals, generating a huge demand of 500 million (2015) vaccine doses every year [2]. Besides egg-based vaccine manufacturing, production platforms based on animal cell culture increasingly contribute to an overall growing market. Thus, the use of suspension MDCK cells (MDCKsus) cultivated in chemically defined medium emerges as a modern vaccine manufacturing platform. In order to improve overall productivity and reduce costs, process analysis, process optimization, and process intensification strategies are necessary. In particular, a better understanding of the effect of virus replication on cell growth, cell morphology and cell metabolism is crucial for developing production processes. In this study, the effect of a synchronous influenza A virus infection on cell growth and central carbon metabolism was investigated. Additionally, intracellular virus replication dynamics of influenza were analyzed and correlated to metabolic pool dynamics. For analysis of intracellular metabolites, an established HPLC-MS method was used to identify and quantify extracted metabolites [3]. A mathematical model, established for adherent MDCK cells, was modified to describe cell growth, consumption and production of main extracellular metabolites [4] as well as dynamics of intracellular metabolite pools of glycolysis and TCA. Our results showed fast infection (\u3c 2 h) of the whole MDCKsus population under the used infection conditions. Intracellular infection was very similar to the already reported dynamics in adherent MDCK cells [5]. Virus particles were released six hours post infection (hpi) for 30 h, with an overall yield of 10,000 virus particles per cell. Despite massively impaired cell growth at 6 hpi, the concentrations of extracellular metabolites did not differ significantly from mock-infected cells used as a control. The majority of intracellular TCA metabolites also followed a similar dynamics. For glycolysis, however, metabolite pools of lower glycolysis decreased rapidly after infection, whereas glucose-6-P and fructose-6-P pools where maintained at a similar level as controls. Overall it seems that influenza infected MDCK cells show primarily an alteration in the glycolysis pathway, channeling metabolites not to the lower part of glycolysis but to the pentose phosphate pathway. Energy metabolism (ATP pools and energy charge) and TCA pools seemed not be affected by virus infection. Quantitative data for mock-infected cells are described by the mathematical model. Work is in progress to explain the dynamics observed in infected cells. [1] Influenza (seasonal) fact sheet (Nov 2016). WHO [online] www.who.int/mediacentre/factsheets/fs211/en/ [2] Palache A. et al., Vaccine 35 (2017): 4681–4686. doi: 10.1016/j.vaccine.2017.07.053 [3] Ritter J.B. et al., Journal of Chrom B, 843 (2006): 216–226. doi: 10.1016/j.jchromb.2006.06.004 [4] Rehberg M. et al., PLoS Comput Biol 10.10 (2014): e1003885. doi: 10.1371/journal.pcbi.1003885 [5] Frensing T. et al., Appl Microbiol Biotechnol 100 (2016): 7181–7192. doi: 10.1007/s00253-016-7542-

Hollow fiber-based high-cell-density and two-stage bioreactor continuous cultivation: Options and limits towards process intensification for virus production

Availability of suspension cell lines and culture media for expansion of up to 20×106 cells/mL provide perfect starting points to develop process intensification strategies for vaccine production. Modern hollow fiber-based perfusion systems accomplish up to 500 ×106 cells/mL in CHO cell cultivations. Reaching 10 to 20 fold higher cell concentrations, while keeping cell specific virus yields constant, could make processes with very low cell specific virus yields (10-100 viruses/cell) already to feasible processes. Therefore, all possible process strategies using new media, cell lines and reactor equipment need revisiting. Data obtained from the production of the modified vaccinia Ankara virus strain MVA-CR19 as well as influenza A/PR/8 virus in either hollow fiber-based high-cell-density (HCD) cultivations (using an alternating tangential flow (ATF) perfusion system) or in two-stage bioreactor continuous cultivations of the suspension cell line AGE1.CR.pIX are presented and critically discussed. Options and limits are highlighted to allow an evaluation of both approaches with respect to scale-up and application to other virus-host cell systems. Both process strategies were successfully scaled-down into shaker flasks allowing parallel experiments. Accordingly, perfusion and semi-perfusion at a feeding rate of 0.05 nL/cell×d led to concentrations of AGE1.CR.pIX cells above 60×106 cells/mL with neither limitation nor overload of nutrients. For infections in 50 mL, a combined strategy comprising an initial fed-batch phase followed by a periodic virus harvest phase resulted in the highest product concentration. Compared to a conventional batch process at 4 to 8×106 cell/mL, maximum titer increased more than 10-fold. Additionally, a 3-fold increase in both cell-specific yield (virus/cell) and volumetric productivity (virus/L×d) could be obtained. The subsequent scale-up into a 1 L bioreactor with ATF perfusion was equally successful and besides allowed re-evaluation of hollow-fiber cut-off. Alternatively, a small scale semi-continuous two-stage cultivation system (100 mL scale, two shaker flasks) was established as an approximation for a genuine continuous bioreactor set-up (1 L scale, two-stage stirred tank bioreactor). MVA virus production at both scales resulted in stable titers of MVA-CR19 virus (approx. 1×108 IU/mL) for over 18 days suggesting an absence of the “von Magnus effect” compared to influenza virus. PCR analysis confirmed stable maintenance of the recombinant transgene in a MVA-CR19.GFP virus. Such a system may be of interest for continuous production of recombinant MVA-based vaccines and gene therapy vectors in the future. Please click Additional Files below to see the full abstract

The EB66® cell line for yellow fever vaccine production at high cell concentrations

The global threat of the emerging yellow fever disease can be effectively countered by vaccination. First vaccines against yellow fever have been developed in embryonated chicken eggs in the 1930s and this production platform remained almost unchanged until today. However, recent outbreaks revealed vaccine supply shortages due to limiting options to ramp up production. Here, we present a cell culture-based process using EB66® cells for production of a live, attenuated yellow fever vaccine (YFV; WHO 17D-213/77 strain). The duck embryo-derived EB66® suspension cell line showed good growth performance in batch mode achieving up to 1.8 × 107 cells/mL and doubling times of less than 17 h in shake flasks in the chemically-defined CDM4Avian medium at 37°C. The seed virus material was adapted by five serial passages to the cell substrate, which resulted in an 8-fold increase in virus titer to 1.3 × 108 PFU/mL (infectious virions per mL). Changes in process temperature and cell disruption to facilitate virus release did not improve final virus titers. In a next step, the process was transferred into benchtop bioreactors equipped with an alternating tangential flow filtration unit (ATF2) operating at a working volume of 700 mL. An on-line conductivity probe was implemented, which enabled cell growth monitoring in real-time. This setup allowed to achieve high cell densities of up to 9.5 × 107 cells/mL resulting in a further increase of YFV titers up to 7.3 × 108 PFU/mL. Based on an input of 4.7 log infectious units per dose, raw virus material equivalent to 10 Mio vaccine doses was produced in less than two weeks operation time. Taken together, EB66® suspension cells can grow to very high cell densities in perfusion systems. Present process intensification clearly demonstrated the potential to produce millions of YFV vaccine doses from small scale cultures in a controllable and scalable manner

Flavivirus production in perfusion processes using the EB66® cell line

The outbreak of mosquito-borne yellow fever virus (YFV) in Angola 2016 rapidly spread to urban regions and other countries. Vaccination campaigns were subsequently intensified, but the increased vaccine demand led to depleted stockpiles. Current yellow fever vaccine manufacturing processes rely on embryonated chicken eggs, which are strongly limited with respect to flexible capacity increase in emergencies. The global vaccine demand is estimated by the WHO to 1.38 billion doses needed to eliminate epidemics. Thus, an urgent need for an improved production platform is needed, ideally transferable to new vaccine developments against emerging flaviviruses, such as Zika virus. Here we present a cell culture-based YFV 17D and Zika virus (ZIKV) production process using the EB66® cell line. The avian EB66® suspension cell line grew fast and stable in chemically defined medium to cell concentrations of 1.8 × 107 cells/mL in shake flasks and batch mode. Seed virus was prepared from Vero-derived YFV and ZIKV material over five passages in EB66® cells. Thereby, infectious virus titers successfully increased by one log unit and maximum titers of 1.4×108 PFU/mL (infectious virions per mL) and 8.0×107 PFU/mL were obtained two days post infection for YFV and ZIKV, respectively. The process was intensified using perfusion bioreactors to increase cell concentrations. Therefore, EB66® cells were cultivated in 1 L benchtop bioreactors equipped with an alternating tangential flow filtration (ATF2) perfusion unit. Perfusion rates were adjusted to maintain glutamine concentrations above 1 mM and cells grew up to 9.5×107 cells/mL. A maximum YFV titer of 7.3 × 108 PFU/mL was achieved. The cell-specific virus yield (CSVY) was 8 PFU/cell, similar to shake flask experiments. For ZIKV production, another approach aimed at the use of on-line capacitance sensors to control cell-specific perfusion rates (CSPRs) based on cell concentrations. This automated system was set to a CSPR of 0.017 and 0.034 nL/cell/day leading to maximum cell concentrations of 8.9×107 cells/mL and 1.6×108 cells/mL. ZIKV titers peaked after three to four days post infection with 2.6×109 PFU/mL and 1.0×1010 PFU/mL, respectively. CSVYs increased from 5 PFU/cell (shake flask experiments) to 30 PFU/cell and even above 64 PFU/cell in this set-up. The increased CSPR resulted in an improved volumetric productivity by factor three compared to the lower CSPR. Further process intensification was achieved by direct cell inoculation to the ZIKV production bioreactor. A 15 mL cryo-bag was thawed with 8.5×108 cells and cell viabilities of 90% after inoculation quickly increased over the cultivation period. Taken together, EB66® suspension cells can be grown to concentrations exceeding 1.5×108 cells/mL in perfusion bioreactors, and cells are highly permissive for YFV and ZIKV. YFV production using perfusion systems generated virus material equivalent to 10 Mio vaccine doses (4.7 log infectious units per dose) in less than two weeks operation time. With the use of on-line sensors to adjust CSPRs meeting cellular nutrient demands, ZIKV titers exceeding 1.0×1010 PFU/mL were obtained for the first time. Direct cryo-bag inoculation shortened the seed train phase, and virus production was initiated with full flexibility. This is a powerful demonstration on how next generation flavivirus vaccine production can be realized