Efficient and stable production of Modified Vaccinia Ankara virus in two-stage semi-continuous and in continuous stirred tank cultivation systems

Abstract

One important aim in cell culture-based viral vaccine and vector production is the implementation of continuous processes. Such a development has the potential to reduce costs of vaccine manufacturing as volumetric productivity is increased and the manufacturing footprint is reduced. In this work, continuous production of Modified Vaccinia Ankara (MVA) virus was investigated. First, a semi-continuous two-stage cultivation system consisting of two shaker flasks in series was established as a small-scale approach. Cultures of the avian AGE1.CR.pIX cell line were expanded in the first shaker, and MVA virus was propagated and harvested in the second shaker over a period of 8–15 days. A total of nine small-scale cultivations were performed to investigate the impact of process parameters on virus yields. Harvest volumes of 0.7–1 L with maximum TCID50 titers of up to 1.0×109 virions/mL were obtained. Genetic analysis of control experiments using a recombinant MVA virus containing green-fluorescent-protein suggested that the virus was stable over at least 16 d of cultivation. In addition, a decrease or fluctuation of infectious units that may indicate an excessive accumulation of defective interfering particles was not observed. The process was automated in a two-stage continuous system comprising two connected 1 L stirred tank bioreactors. Stable MVA virus titers, and a total production volume of 7.1 L with an average TCID50 titer of 9×107 virions/mL was achieved. Because titers were at the lower range of the shake flask cultivations potential for further process optimization at large scale will be discussed. Overall, MVA virus was efficiently produced in continuous and semi-continuous cultivations making two-stage stirred tank bioreactor systems a promising platform for industrial production of MVA-derived recombinant vaccines and viral vectors.

Continuous influenza virus production in a tubular bioreactor system provides stable titers and avoids the “von Magnus effect”

“…Batch cultures were initiated with cells of the CSTR of the continuous tubular bioreactor system (see Fig 1) after termination of continuous cultivations. B) Influenza HA titers (closed diamonds) and TCID 50 titers (open symbols) of a semi-continuous culture performed in shake flasks as described previously [6].…”

“…An MOI of 0.025 was used. The methodology for sampling, medium exchanges and harvesting, as well as the equations required for operating this semi-continuous cultivation system is described in detail in [6]. HA titers, TCID 50 titers, and segment-specific PCR were analyzed.…”

“…Despite small differences in process operation and parameters among these platforms, all processes are basically operated in batch mode. Moving from batch to continuous production could significantly improve volumetric productivity (virus produced/[(time)×(volume of cultured media used)]) and reduce the manufacturing footprint [6]. Continuous production is currently not only promoted by various manufacturers of recombinant CHO cell-based biologicals, but also by regulatory agencies [7].…”

“…Cascades of stirred tank bioreactors have been used since the 1960’s for production of viruses in continuous mode [8]. This included adenovirus, poliovirus, baculovirus, picornavirus [9], influenza virus [10], and Modified Vaccinia Ankara virus [6]. The cascades are characterized by one continuous stirred tank reactor (CSTR) for cell growth and at least one CSTR in series for virus infection and propagation.…”

A Deleted Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara (MVA)

“…A high dose of 10 8 infectious units are estimated to be required for vaccination because MVA is not amplified (replicated) at the injection site (Wyatt et al 2004). Production of MVA poses certain challenges (Cottingham and Carroll 2013) that may be alleviated with a new strain such as MVA-CR that would allow simplified processes or even continuous production in true suspension cultures (Tapia et al 2017;Vázquez-Ramírez et al 2018). We have now characterized MVA-CR19 in greater detail and our results indicate that the genomic structure of this strain deviates from wildtype virus in more aspects than we had previously anticipated.…”