Combining cognitive and system-oriented approaches for designing IR user interfaces

Poster at the AIR workshop 2008, London, Englan

Discovery and molecular characterization of West Nile virus NY 1999

An account of the 1999 outbreak of West Nile virus infections in New York City

Discovery and molecular characterization of West Nile virus NY 1999

An account of the 1999 outbreak of West Nile virus infections in New York City

Continuous production of viral vaccines with a two-stage bioreactor system

Continuous processes can be particularly efficient for production of biologicals that are required in large amounts such as viral vaccines. One virus that has received much clinical attention is Modified Vaccinia Ankara virus (MVA), which is a potential platform for expression of recombinant viral antigens and can be used as a vector in gene therapy [1]. Recently, a new MVA virus strain has been successfully propagated at high yields in non-aggregated avian suspension cells [2] allowing the production of MVA virus in continuous bioreactors. MVA is a lytic DNA virus and therefore, continuous production strategies can be implemented using two-stage bioreactor systems, 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, continuous production of MVA virus in a two-stage bioreactor (TSB) set-up (two 1 L stirred tank bioreactors) was evaluated. Subsequently, the set-up was scaled down to a non-instrumented semi-continuous cultivation system (two shaker flasks; small-scale culture, SSC) as approximation to a continuous cultivation [4] that would facilitate TSB screening. The virus strain MVA.CR19 and the duck cell line AGE1.CR.pIX (both from ProBioGen, Berlin) were used. The TSB system involved a bioreactor for cell growth and a second bioreactor in series for virus propagation [3]. The SSC system consisted of two shaker flasks, one for cell growth (120 mL working volume) and another for virus propagation (different residence times). Harvest, cell transfer, and addition of fresh medium were done manually twice a day. Continuous production of MVA-CR19 was maintained for 18 d with the TSB system. Virus titers showed 7 d of transient phase, followed by stable titers that suggested the absence of a Von Magnus effect over 18 d. A total production capacity of 2x1010 viruses/day was estimated (4x1010 viruses/day estimated for batch). The space-time yield of the TSB approached that of 2 parallel batches at 11 d post infection. The process was scaled down to the SSC system that resulted in stable production of cells, and virus titers that approached the dynamics and values obtained with the TSB system. Additional cultivations with the SSC system showed that different residence times in the virus bioreactor could influence virus titers. Overall, it was demonstrated that continuous production of MVA.CR19 virus in a TSB system is feasible. Also, a small scale two-stage semi-continuous cultivation was successfully established as a faster and cheaper tool for screening the TSB systems before scale-up

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

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

Ribavirin inhibits West Nile virus replication and cytopathic effect in neural cells

West Nile virus (WNV) is an emerging mosquito-borne pathogen that was reported for the first time in the Western hemisphere in August 1999, when an encephalitis outbreak in New York City resulted in 62 clinical cases and 7 deaths. WNV, for which no antiviral therapy has been described, was recently recovered from a pool of mosquitoes collected in New York City. In anticipation of the recurrence of WNV during the summer of 2000, an analysis was made of the efficacy of the nucleoside analogue ribavirin, a broad-spectrum antiviral compound with activity against several RNA viruses, for treatment of WNV infection. High doses of ribavirin were found to inhibit WNV replication and cytopathogenicity in human neural cells in vitro

Intensification of MVA and influenza virus production through high-cell-density cultivation approaches

Background. Unlike production of recombinant proteins, continuous production of viral vaccines at high cell densities (HCD) is often constrained by a decrease in cell-specific virus yields, early host cell lysis during virus propagation and limited virus recovery from culture broth. Nevertheless, advanced fed-batch [1] and perfusion strategies can be applied to achieve high-yield virus production processes. In this study, the development of a semi-continuous process for the production of the modified vaccinia Ankara virus isolate MVA-CR19 and influenza virus A/PR/8/34 (H1N1) in HCD cultivations of the suspension cell line AGE1.CR.pIX (ProBioGen AG, Berlin) is presented. Methods. Depending on the required scale, high cell concentrations (~ 50×106 cells/mL) were achieved either through medium renewal by periodic centrifugation (semi-perfusion) in 50 mL cultivations or using an alternating tangential flow (ATF) perfusion system for 1 L bioreactors. Process development and optimization comprised three phases: 1) assessment of different fed-batch and medium exchange strategies for the propagation of MVA-CR19 or influenza A/PR/8/34 viruses in 50 mL cultivations; 2) scale-up and process optimization of the selected high-yield process strategy to a 1 L bioreactor with the ATF system, and 3) integration of a one-step purification process using magnetic sulfated cellulose particles (MSCP). For both viruses, conventional batch cultivation (no addition/medium exchange after infection) was compared with processes applying fed-batch, periodic medium exchange and the combination of both during virus propagation. Results. Perfusion and semi-perfusion at a feeding rate of 0.05 nL/cell×d was suitable to propagate AGE1.CR.pIX cells above 60×106 cells/mL with neither limitation nor overload of nutrients. For infections at 50 mL scale, the application of a combined strategy comprising an initial fed-batch phase followed by a periodic virus harvest phase resulted in the highest product yield with a more than 10-fold increase in virus particles concentration compared to the conventional batch processes operated at 4 to 8×106 cells/mL [2]. Additionally, a 3-fold increase in both cell-specific yield (virus particles/cell) and volumetric productivity (virus particles/L×d) could be obtained. Comparable yields were observed when up-scaling to a 1 L bioreactor using an ATF-system, even when virus particles were retained within the bioreactor. Further selection of the optimal pore size of the ATF membrane allowed semi-continuous harvesting of the produced viruses and its purification with MSCPs with a recovery from 30 to 50%. In all cases, cell-specific yields and volumetric productivities reached their maxima at 72 h post-infection, indicating that the process should be stopped at that time point. Conclusion. Compared to conventional batch processes, the developed HCD process offers significantly higher productivities including the option to integrate a one-step purification process in a semi-continuous mode. Overall, the results show that there is a great potential for semi-continuous HCD processes for the production of viral vaccines in larger scales, which could support efforts towards the establishment of continuous vaccine manufacturing. References. 1. Pohlscheidt, M., et al., 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. Vaccine, 2008. 26(12): p. 1552-65. 2. Lohr, V., et al., New avian suspension cell lines provide production of influenza virus and MVA in serum-free media: studies on growth, metabolism and virus propagation. Vaccine, 2009. 27(36): p. 4975-82