Abstract
Process intensification and integration is crucial regarding an ever increasing pressure on manufacturing costs and capacities in biologics manufacturing. For virus production in perfusion mode, membrane-based alternating tangential flow filtration (ATF) and acoustic settler are the commonly described cell retention technologies. While acoustic settlers allow for continuous influenza virus harvesting, the use of commercially available membranes for ATF systems typically results in the accumulation of virus particles in the bioreactor vessel. Accordingly, with one single harvest at the end of a cultivation, this increases the risk of lowering the product quality. To assess which cell retention device would be most suitable for influenza A virus production, we compared various key performance figures using AGE1.CR.pIX cells at concentrations between 25 and 50 × 106 cells/mL at similar infection conditions using either an ATF system or an acoustic settler. Production yields, process-related impurities, and aggregation of viruses and other large molecules were evaluated. Taking into account the total number of virions from both the bioreactor and the harvest vessel, a 1.5–3.0-fold higher volumetric virus yield was obtained for the acoustic settler. In addition, fewer large-sized aggregates (virus particles and other molecules) were observed in the harvest taken directly from the bioreactor. In contrast, similar levels of process-related impurities (host cell dsDNA, total protein) were obtained in the harvest for both retention systems. Overall, a clear advantage was observed for continuous virus harvesting after the acoustic settler operation mode was optimized. This development may also allow direct integration of subsequent downstream processing steps.Key points• High suspension cell density, immortalized avian cell line, influenza vaccine.
Highlights
Universitätsplatz 2, 39106 Magdeburg, GermanyEver increasing demands for vaccination and gene therapy have raised the need to develop efficient large-scale manufacturing processes, including cell culture–based virus production (Kaemmerer 2018; Rappuoli and Hanon 2018)
While recombinant protein production in perfusion mode has been established in the industry for many years (Konstantinov and Cooney 2015), virus production in perfusion mode is mainly pursued in academic research (Gutiérrez-Granados et al 2018; Tapia et al 2016)
In order to assess the impact of the cell retention device and the recirculation strategy on the influenza virus production, perfusion cell cultures using similar infection conditions but with different recirculation strategies and recirculation flow rates were carried out
Summary
Universitätsplatz 2, 39106 Magdeburg, GermanyEver increasing demands for vaccination and gene therapy have raised the need to develop efficient large-scale manufacturing processes, including cell culture–based virus production (Kaemmerer 2018; Rappuoli and Hanon 2018). While recombinant protein production in perfusion mode has been established in the industry for many years (Konstantinov and Cooney 2015), virus production in perfusion mode is mainly pursued in academic research (Gutiérrez-Granados et al 2018; Tapia et al 2016). Viruses such as influenza virus (Genzel et al 2014; Petiot et al 2011), attenuated yellow fever virus (Nikolay et al 2018), adenovirus (Henry et al 2004), lentivirus (Manceur et al 2017), and modified vaccinia Ankara virus In case of influenza pandemics, intensified cell culture–based perfusion cultures allowing a rapid and small footprint production of viruses could be of particular interest (Hegde 2015)
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