High titer MVA and influenza A virus production using a hybrid fed-batch/perfusion strategy with an ATF system

Abstract

A cultivation strategy to increase the productivity of Modified Vaccinia Ankara (MVA) virus in high-cell density processes is presented. Based on an approach developed in shake flask cultures, this strategy was established in benchtop bioreactors, comprising the growth of suspension AGE1.CR.pIX cells to high cell densities in a chemically defined medium before infection with the MVA-CR19 virus strain. First, a perfusion regime was established to optimize the cell growth phase. Second, a fed-batch regime was chosen for the initial infection phase to facilitate virus uptake and cell-to-cell spreading. Afterwards, a switch to perfusion enabled the continuous supply of nutrients for the late stages of virus propagation. With maximum infectious titers of 1.0 × 10 10 IU/mL, this hybrid fed-batch/perfusion strategy increased product titers by almost one order of magnitude compared to conventional batch cultivations. Finally, this strategy was also applied to the production of influenza A/PR/8/34 (H1N1) virus considered for manufacturing of inactivated vaccines. Using the same culture system, a total number of 3.8 × 10 10 virions/mL was achieved. Overall, comparable or even higher cell-specific virus yields and volumetric productivities were obtained using the same cultivation systems as for the conventional batch cultivations. In addition, most viral particles were found in the culture supernatant, which can simplify further downstream operations, in particular for MVA viruses. Considering the current availability of well-described perfusion/cell retention technologies, the present strategy may contribute to the development of new approaches for viral vaccine production. Electronic supplementary material The online version of this article (10.1007/s00253-019-09694-2) contains supplementary material, which is available to authorized users.

Performance of an acoustic settler versus a hollow fiber–based ATF technology for influenza virus production in perfusion

“…Similar doubling times and high viabilities before infection were also observed for the cell growth phase in the present study comparing an acoustic settler with different recirculation strategies and an ATF system. Furthermore, the t d values obtained with the acoustic settler in this study were in agreement with values reported previously for AGE1.CR.pIX cells cultivated in perfusion mode with an ATF system operated at higher exchange rates (between 28 and 41 h) Vazquez-Ramirez et al 2019).…”

“…1A) with a polyethersulfone (PES) hollow fiber membrane (0.2 μm pore size, 470 cm 2 , 76 fibers with 0.9 mm diameter, Spectrum). The recirculation exchange flow rate was decreased from 1.0 to 0.8 L/min compared with previous studies (Genzel et al 2014;Vazquez-Ramirez et al 2019) to ensure a laminar flow regime in the membranes (see "Hydrodynamic stress" section). (2) An acoustic settler (10 L acoustic chamber version, SonoSep Technologies) ( Fig.…”

“…Efficient perfusion cultures using AGE1.CR.pIX cells, which are characterized by short doubling times (t d ), high viabilities, and high cell concentrations (up to 50 × 10 6 cells/mL), were described previously for cultivations with bioreactors coupled to an ATF device Genzel et al 2014;Vazquez-Ramirez et al 2018;Vazquez-Ramirez et al 2019). Before evaluating virus yields and productivity, the growth performance of AGE1.CR.pIX cells was assessed for VCCs between 25 and 55 × 10 6 cells/mL using an acoustic settler.…”

“…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 (Vazquez-Ramirez et al 2019) have already been investigated for intensified production in perfusion culture. Using different setups, this technology has shown clear advantages, such as a higher volumetric productivity compared with batch processes.…”

“…High cell concentrations further confound the mentioned factors affecting product sieving. Membrane clogging (Cortin et al 2004) as well as unwanted virus accumulation inside the bioreactor (Genzel et al 2014;Vazquez-Ramirez et al 2018;Vazquez-Ramirez et al 2019) have been reported for membrane-based perfusion processes. In contrast, continuous harvesting of recombinant proteins has been shown to improve cell-specific productivity and product quality due to a shorter residence time inside the bioreactor (Patil and Walther 2018).…”