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. Because this vector is unable to replicate in human recipients, a high amount of virions per dose is required 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 we compared different options for cell retention in perfusion mode using conventional stirred‐tank bioreactors including an alternating tangential flow filtration system, an acoustic settler and an inclined settler. The last two options allowed continuous MVA virus harvesting. Furthermore, we studied hollow‐fiber based bioreactors and an orbital‐shaken bioreactor in perfusion mode, both available for single‐use. Productivity for the virus strain MVA‐CR19 was compared to results from batch and continuous production reported in literature. Our results demonstrate that MVA virus is highly stable at 37°C in cell culture so that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred‐tank bioreactor, a perfusion strategy during the whole run with working volume expansion after virus infection resulted in the highest yields. Overall, infectious MVA virus titers of 2.1–16.5 × 10 9 virions/mL were 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. This article is protected by copyright. All rights reserved

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis

“…An acoustic settler device with a power of 2 W and a frequency of 2.1 MHz was used for cell retention. The parameters of the acoustic settler were set as reported in a previous publication (Gränicher et al, 2020). A constant recirculation flow rate of five reactor volumes per day (day −1 ) was applied for the acoustic settler system.…”

“…Samples purified by SXC were sonicated with a VialTweeter (UP200St, Power = 160 W, Amplitude = 100%, Pulse = 30%; Hielscher Ultrasound Technology) to dissolve virus aggregates before measurements. The total number of infectious virions measured in the harvest vessel and in the bioreactor (Vir tot , based on TCID 50 ), the concentration of infectious virions produced (C vir, tot , TCID 50 /ml), the volumetric virus productivity (P v , TCID 50 /L/day), and the cell‐specific infectious virus yield (CSVY, TCID 50 /cell) were calculated as described previously by Gränicher et al (2020). The STY of purified MVA was calculated based on the bioreactor V w (TCID 50 /L bioreactor /day; similar calculation compared with P v , but replacing the spent cell culture medium volume with V w ).…”

“…Intensification can be achieved with bioreactors coupled to devices for harvesting of infectious units with subsequent continuous purification. A techno‐economic analysis could provide insights about cost differences between a batch and a perfusion process for viral vector production (Cameau et al, 2019; Gränicher et al, 2020; Pearson, 2020), similarly to recombinant protein production (Klutz et al, 2016; Lim et al, 2006; Pleitt et al, 2019; Pollock et al, 2013). This could allow identifying key factors and bottlenecks allowing cost‐savings.…”

Cell culture–based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

“…Besides membrane-based cell retention devices, there also exist other systems. The potential of two of these systems (acoustic settler and inclined settler) for continuous virus harvesting was described recently for IAV and MVA production (Coronel et al 2020;Granicher et al 2020Granicher et al , 2021. Choosing one of these systems will depend on many parameters and a detailed comparison of pros and cons can be found elsewhere (Chotteau 2015).…”

Biomanufacturing of Gene Therapy Vectors

“…Further improvements in the reactor production are achieved by the establishment of high cell density culture systems as shown for the baculovirus/Sf9 system for the production of AAV under optimized conditions (10 × more cells produce also ten times more vector amount) [ 3 ] or for MVA production under perfusion conditions (Gränicher et al. [ 5 ] —comparison of different perfusion systems).…”