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

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

Forced Degradation Testing as Complementary Tool for Biosimilarity Assessment

Oxidation of monoclonal antibodies (mAbs) can impact their efficacy and may therefore represent critical quality attributes (CQA) that require evaluation. To complement classical CQA, bevacizumab and infliximab were subjected to oxidative stress by H2O2 for 24, 48, or 72 h to probe their oxidation susceptibility. For investigation, a middle-up approach was used utilizing liquid chromatography hyphenated with mass spectrometry (LC-QTOF-MS). In both mAbs, the Fc/2 subunit was completely oxidized. Additional oxidations were found in the light chain (LC) and in the Fd’ subunit of infliximab, but not in bevacizumab. By direct comparison of methionine positions, the oxidized residues in infliximab were assigned to M55 in LC and M18 in Fd’. The forced oxidation approach was further exploited for comparison of respective biosimilar products. Both for bevacizumab and infliximab, comparison of posttranslational modification profiles demonstrated high similarity of the unstressed reference product (RP) and the biosimilar (BS). However, for bevacizumab, comparison after forced oxidation revealed a higher susceptibility of the BS compared to the RP. It may thus be considered a useful tool for biopharmaceutical engineering, biosimilarity assessment, as well as for quality control of protein drugs

A new porcine suspension cell line (PBG.PK-21) provides efficient production for influenza and yellow fever vaccine viruses

Shifting from egg-based influenza and yellow fever vaccine production towards efficient cell culture-based manufacturing is one of the biggest challenges in viral vaccine manufacturing. Innovative bioprocesses, such as high cell density [1] or continuous virus cultivations [2] improving cell-based viral vaccine production have been shown over the past years. However, finding the right high-yield cell substrate is still pending for many important viruses. Ideally, such host cell lines should enable the efficient production of several virus strains to set-up manufacturing platforms. Here, we report on a novel suspension cell line (PBG.PK-21 (ProBioGen) derived from immortal porcine kidney cells. These cells were first adapted to chemically defined virus production medium CD-U5 (ProBioGen) and agitated suspension culture. Despite its robust growth these cells were not yet suitable for virus manufacture due to a chronic infection with porcine circovirus 1 (PCV1) that is often found in porcine cell lines. The cells were cured from infection after siRNA mediated suppression of the contaminating virus followed by single cell cloning. Clone number 21 proven to be free from PCV1 over multiple passages and distinguished by high peak cell densities and good productivity for other viruses was chosen for further analysis. Cell growth, cell metabolism and virus production were characterized in shake flasks and bioreactors. Cell concentration up to 8 x 106 cells/mL and a doubling time of 33 h were obtained in batch mode in CD-U5 medium. Furthermore, process intensification using either semi-perfusion in shake flasks or hollow fiber-based perfusion with an ATF2 system coupled to a 0.6 L wv stirred tank bioreactor system was evaluated. In this system, cell densities up to 42 x 106 cells/mL were achieved with a cell-specific perfusion rate of 0.07 nL/cell/day. After optimization of influenza A/PR/8/34 (H1N1) virus production, HA titres of 3.3 log(HAU/100µL) with a cell-specific yield of 5200 virions/cells were achieved in bioreactor fed-batch mode. The PBG.PK-21 suspension cell line also shows potential to be used in a modern cell-based swine influenza vaccine production process for the veterinary market. Indeed, similar competitive virus titres (3.4 log(HAU/100µL)) and cell-specific yields were found for the swine influenza A/Bakum/1832/00 (H1N2) virus in batch mode. Finally, tests were also conducted regarding yellow fever virus production (live-attenuated 17DD YFV, RKI). In first scouting experiments, promising titres up to 3 x 106 PFU/mL were obtained in batch mode. First attempts towards process intensification using ATF-based perfusion systems with manually-adapted perfusion rate showed successful maintenance of cell-specific influenza A virus yields at high cell density. Titres up to 4 log(HAU/100µL) were obtained with 42 x 106 cells/mL. The glycosylation profile of influenza A/PR8/34 produced in PBG.PK-21 cell line was also analysed and will be compared to profiles of other cell lines. Overall, PBG.PK-21 suspension cells show a great potential to become the cell substrate of choice for many existing and new viral vaccine processes in next generation high-yield human as well as veterinary vaccine manufacturing. [1] Genzel, Y., et al., Vaccine, 2014. 32(24): p. 2770-81. [2] Tapia F, et al., PLoS ONE, 2017, 12(8): e0182553

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