Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production

Abstract

Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers, requiring a high amount of virions per dose for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA-CR19 is an option to obtain very high MVA yields. Here the authors compare different options for cell retention in perfusion mode using conventional stirred-tank bioreactors. Furthermore, the authors study hollow-fiber bioreactors and an orbital-shaken bioreactor in perfusion mode, both available for single-use. Productivity for the virus strain MVA-CR19 is compared to results from batch and continuous production reported in literature. The results demonstrate that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred-tank bioreactor, a perfusion strategy with working volume expansion after virus infection results in the highest yields. Overall, infectious MVA virus titers of 2.1-16.5 × 10⁹ virions/mL are achieved in these intensified processes. Taken together, the study shows a novel perspective on high-yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single-use perfusion platforms.

Keywords: Modified Vaccinia Ankara virus; continuous production; perfusion cell culture; process intensification.

Figure 1

Simplified scheme of process options considered for yield comparisons towards establishment of a platform technology for MVA virus production. Green arrows indicate the continuous or semi‐continuous addition of fresh medium during cultivation. Red arrows indicate continuous or semi‐continuous removal of cell culture broth containing either only medium, medium with cell debris, medium with cells or—during virus production phase—medium with MVA virus or medium with cells, cell debris, and MVA virus. Thick blue arrows indicate stepwise process development performed in our group.

Figure 2

Perfusion mode cultivations in stirred‐tank bioreactor for MVA‐CR19.GFP virus production in AGE1.CR.pIX cells at high cell density. A) Viable cell concentration (●), B) cell viability (●), C) cell metabolites (glucose (●), lactate (▲), and ammonium (■)), D) concentration of virions produced (●), E) cell‐specific virus yield, and F) volumetric virus productivity of AGE1.CR.pIX cells for run 1 (ATF, black), run 2 (acoustic settler, red), run 3 (acoustic settler, orange), and run 4 (inclined settler, blue). Process parameters as described in Sections 4 and 2.1. No data available for run 3, graph C. The vertical dotted lines correspond to the time of infection.

Figure 3

Perfusion mode cultivations in single‐use orbital‐shaken bioreactor with ATF (OSB‐ATF) and hollow‐fiber bioreactor (HFBR) for MVA‐CR19 virus production in AGE1.CR.pIX cells at high cell density. A) Viable cell concentration (●), B) concentration of virions produced (●), C) cell‐specific virus yield, and D) volumetric virus productivity for the production of MVA‐CR19 virus for run 5 (black, OSB‐ATF in hybrid perfusion mode), run 6 (red, HFBR in perfusion mode), run 7 (orange, HFBR in perfusion mode), run 8 (dark blue, stirred‐tank bioreactor with ATF in hybrid perfusion mode, control experiment, run hybrid 1 from Vazquez et al. (2019)^{[ 27 ]}), and run 9 (light blue, stirred‐tank bioreactor with ATF in hybrid perfusion mode, control experiment, run hybrid 2 from Vazquez et al. (2019)^{[ 27 ]}). For run 6 and 7, cell concentrations given are from samples taken from the extra‐capillary space when medium was exchanged. However, as many cells were attached to the hollow‐fiber the overall cell concentrations might be higher. For the three last sampling points of run 6 and the last sampling point of run 7, the HFBR was flushed thoroughly to detach all cells. The vertical dotted lines correspond to the time of infection.

Figure 4

MVA virus production in batch, perfusion, hybrid perfusion or continuous mode. A) Maximum concentration of virions produced over a period of 30 days and B) over a period of 90 days. C) Total number of virions produced considering the maximum production scale for single‐use operation. D) Total number of cells present during the virus production phase considering the maximum production scale for single‐use operation. For graph D, the hollow‐fiber bioreactor (HFBR) based process was not taken into account as the monitoring of cell concentrations was difficult (Section 4). Each step corresponds to one independent run. Batch (black, average of four runs, data not shown), perfusion (grey, average of runs 1–4), pseudo hybrid perfusion (dotted red, average of three runs, data not shown), hybrid perfusion (red, average of runs 8–9), HFBR (blue, average of runs 6–7), two‐stage semi‐continuous shake‐flask cultivation system (dotted green, average of runs SM25‐A and SM25‐B^{[ 32 ]}) and two‐stage continuous stirred‐tank cultivation system (green, run T25^{[ 32 ]}). For a more accurate estimation, the process time includes 1 day for system set‐up before the start of a cell culture run and 1 day for system clean‐up/disassembly at the end of the run. For graphs C and D, the maximum single‐use production scales assumed were: batch, 6000 L;^{[ 58 ]} perfusion and hybrid perfusion, 3000 L (www.samsungbiologics.com); continuous, 6000 L (cell growth bioreactor); and 3000 L (virus production bioreactor);^{[ 58 ]} HFBR, 2.1 L.^{[ 45} , ^{46 ]}