Process intensification of EB66® cell cultivations leads to high-yield yellow fever and Zika virus production

Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-018-9275-z BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Process intensification of EB66® cell cultivations leads to high-yield yellow fever and Zika virus production Alexander Nikolay 1 & Arnaud Léon 2 & Klaus Schwamborn 2 & Yvonne Genzel 1 & Udo Reichl 1,3 Received: 3 April 2018 / Revised: 24 July 2018 / Accepted: 25 July 2018 # The Author(s) 2018 Abstract A live-attenuated, human vaccine against mosquito-borne yellow fever virus has been available since the 1930s. The vaccine provides long-lasting immunity and consistent mass vaccination campaigns counter viral spread. However, traditional egg-based vaccine manufacturing requires about 12 months and vaccine supplies are chronically close to shortages. In particular, for urban outbreaks, vaccine demand can be covered rarely by global stockpiling. Thus, there is an urgent need for an improved vaccine production platform, ideally transferable to other flaviviruses including Zika virus. Here, we present a proof-of-concept study regarding cell culture-based yellow fever virus 17D (YFV) and wild-type Zika virus (ZIKV) production using duck embryo-derived EB66® cells. Based on comprehensive studies in shake flasks, 1-L bioreactor systems were operated with scalable hollow fiber-based tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) perfusion systems for process intensification. EB66® cells grew in chemically defined medium to cell concentrations of 1.6 × 108 cells/mL. Infection studies with EB66®-adapted virus led to maximum YFV titers of 7.3 × 108 PFU/mL, which corresponds to about 10 million vaccine doses for the bioreactor harvest. For ZIKV, titers of 1.0 × 1010 PFU/mL were achieved. Processes were automated successfully using a capacitance probe to control perfusion rates based on on-line measured cell concentrations. The use of cryo-bags for direct inoculation of production bioreactors facilitates pre-culture preparation contributing to improved process robustness. In conclusion, this platform is a powerful option for next generation cell culture-based flavivirus vaccine manufacturing. Keywords EB66® . Yellow fever virus . Zika virus . Flavivirus . Perfusion . Capacitance probe Introduction Yellow fever virus (YFV) belongs to the arthropod-borne Flavivirus genus circulating between non-human primates in the sylvatic cycle. Repeatedly, transmission vectors like Aedes Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-018-9275-z) contains supplementary material, which is available to authorized users. * Yvonne Genzel 1 Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, Sandtorstr. 1, 39106 Magdeburg, Germany 2 Valneva SE, 6 rue Alain Bombard, 44800 Saint-Herblain, France 3 Otto von Guericke University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany aegypti mosquitos introduce the virus to humans in urban regions causing thousands of deaths and very serious humanitarian consequences (WHO 2016b). The lack of specific therapies for disease treatment turns vaccination into the only preventive countermeasure. Already in 1937, a very effective live-attenuated YFV vaccine was developed and manufactured in embryonated chicken eggs (Theiler and Smith 1937). Since then, the production process remained essentially unchanged through to the present day. However, when vaccination campaigns were augmented during YFV outbreaks in Angola 2016, egg-based production levels could not meet the immediate increase in vaccine demand. As a consequence, dose-sparing practices were applied to stretch vaccine supplies, but the depletion of global emergency stockpiles could not be prevented (Monath et al. 2016). Simultaneously, spreading to China that is now infested with A. aegypti but was so far considered free of YFV was documented (Wilder-Smith and Leong 2017). This underpins the inherent threat to public health and the urgent need to expand Appl Microbiol Biotechnol global YFV vaccine stockpiles (Calisher and Woodall 2016; Vasconcelos and Monath 2016). In total, the WHO estimates the global YFV vaccine demand to 1.38 billion vaccine doses for the next decades to eliminate epidemics (WHO 2016a). However, provision of a safe and fast vaccine supply based on production processes relying exclusively on pathogen-free fertilized hen’s eggs is disputable. In addition, the development of vaccines against other emerging and re-emerging viruses, such as Zika virus (ZIKV), will require additional resources. Accordingly, alternative manufacturing platforms need to be considered. This involves the use of continuous cell lines, like the adherent Vero cell (Diamond and Coyne 2017; Monath et al. 2010). However, anchorage-dependent cell growth poses serious limitations for large-scale vaccine manufacturing and process intensification (Gallo-Ramirez et al. 2015; Genzel and Reichl 2009). In contrast, suspensionadapted cell lines (like PER.C6®, AGE1.CR®, MDCK.SUS, EB66®, CAP®, and BHK-21 cells) showed promising cell growth in bioreactors and productivities for a wide range of viruses (Brown and Mehtali 2010; Chu et al. 2009; Genzel et al. 2013; Jordan et al. 2009; Leon et al. 2016; Lohr et al. 2009; Nikolay et al. 2017; Pau et al. 2001). Here, we present the use of the duck embryo-derived EB66® cells as a substrate for efficient YFV and ZIKV propagation. Hollow fiber-based perfusion processes in bioreactors equipped with an on-line capacitance sensor for perfusion rate control were used to optimize cell growth and increase virus titers. Results clearly demonstrate that this platform is wellsuited for process development and intensification in vaccine manufacturing, particularly for viruses that only replicate at a low cell-specific virus yield (up to 10 infectious virions per cell). Materials and methods Cell lines and viruses EB66® suspension cells (Valneva SE) were initially maintained in EX-CELL EBx GRO-I serum-free medium (SAFC Biosciences) supplemented with 2.5 mM L-glutamine (Sigma) and cultivated in 125-mL non-baffled shake flasks (working volume 45 mL) using an orbital shaker at 37 °C, 7.5% CO2 atmosphere, and 150 rpm with 50-mm shaking diameter (Multitron Pro, Infors HT). For further experiments, cells from a thawed cryo-vial were directly adapted to growth in the chemically defined HyClone CDM4Avian medium (GE Healthcare) supplemented with 2.5 mM L-glutamine and passaged at least three times before performing experiments. Porcine kidney stable (PS) cells (courtesy of M. Niedrig, Robert Koch Institute, Berlin, Germany) were used for the plaque assay and were maintained in Glasgow’s minimum essential medium (GMEM) with 10% (v/v) fetal bovine serum (Gibco), 2% (w/v) FMV Peptone (LaB-M), 2 mM L-glutamine, and 2 mM pyruvate (Sigma). The live-attenuated yellow fever virus (YFV 17D-204, produced in specific pathogen-free eggs) was k (...truncated)