Influenza A virus production in a single-use orbital shaken bioreactor with ATF or TFF perfusion systems

Coronel, Juliana; Behrendt, Ilona; Bürgin, Tim; Anderlei, Tibor; Sandig, Volker; Reichl, Udo; Genzel, Yvonne

doi:10.1016/j.vaccine.2019.06.005

Cited by 30 publications

(45 citation statements)

References 36 publications

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“…After infection, perfusion was interrupted for approximately 1 h to allow for virus entry. In the first experiment, the CSPR was maintained after infection, similarly to previous studies (Genzel et al, 2014;Coronel et al, 2019a), which resulted in perfusion rates of 0.6-2.0 day −1 (IS1) in this case. In the following cultivations, a constant perfusion rate of 2 day −1 was used after infection to allow for a shorter residence time of the virions inside the bioreactor.…”

Section: Perfusion Bioreactor Cultivationssupporting

confidence: 60%

“…Imaging flow cytometry was used to determine the fraction of cells infected with IAV. Samples were processed as described previously (Frensing et al, 2016;Coronel et al, 2019a). Therefore, samples containing 2 × 10 6 cells were collected during virus production phase.…”

Section: Virus Titrationmentioning

confidence: 99%

“…Therefore, samples containing 2 × 10 6 cells were collected during virus production phase. Following fixation with paraformaldehyde, samples were stored in 70% ethanol at −20 • C. Antibody staining for viral nucleoprotein (NP) combined with nuclear staining using DAPI were done according to Coronel et al (2019a), adapted from Frensing et al (2016). In brief, the main steps were blocking at 37 • C for 30 min; incubation with monoclonal mouse anti-NP antibody mAb61A5 [from F. Momose, (Frensing et al, 2016)] diluted 1:500; incubation with AF 647-conjugated polyclonal goat anti-mouse antibody (Life Technologies, A21235) diluted 1:500; addition of DAPI (approximately 5 µg/mL).…”

Section: Virus Titrationmentioning

confidence: 99%

“…In addition, product quality attributes can be improved in perfusion cultures, for example, by reducing product heterogeneity (Bielser et al, 2018), or preventing the accumulation of growth inhibitors and metabolic waste products. Most perfusion studies in lab-scale bioreactors for virus vaccine production were carried out using filtration systems, such as spin-filters (Perrin et al, 1995), tangential flow filtration (TFF;Nikolay et al, 2018;Coronel et al, 2019a), or alternating tangential flow filtration (ATF; Genzel et al, 2014;Vazquez-Ramirez et al, 2018;Gränicher et al, 2019). The latter is the most commonly used perfusion system in recombinant protein production (Bielser et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode

Coronel

Gränicher

Sandig³

et al. 2020

Front. Bioeng. Biotechnol.

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Influenza viruses have been successfully propagated using a variety of animal cell lines in batch, fed-batch, and perfusion culture. For suspension cells, most studies reported on membrane-based cell retention devices typically leading to an accumulation of viruses in the bioreactor in perfusion mode. Aiming at continuous virus harvesting for improved productivities, an inclined settler was evaluated for influenza A virus (IAV) production using the avian suspension cell line AGE1.CR.pIX. Inclined settlers present many advantages as they are scalable, robust, and comply with cGMP regulations, e.g., for recombinant protein manufacturing. Perfusion rates up to 3000 L/day have been reported. In our study, successful growth of AGE1.CR.pIX cells up to 50 × 10 6 cells/mL and a cell retention efficiency exceeding 96% were obtained with the settler cooled to room temperature. No virus retention was observed. A total of 5.4-6.5 × 10 13 virions were produced while a control experiment with an ATF system equaled to 1.9 × 10 13 virions. For infection at 25 × 10 6 cells/mL, cell-specific virus yields up to 3474 virions/cell were obtained, about 5-fold higher than for an ATF based cultivation performed as a control (723 virions/cell). Trypsin activity was shown to have a large impact on cell growth dynamics after infection following the cell retention device, especially at a cell concentration of 50 × 10 6 cells/mL. Further control experiments performed with an acoustic settler showed that virus production was improved with a heat exchanger of the inclined settler operated at 27 • C. In summary, cell culturebased production of viruses in perfusion mode with an inclined settler and continuous harvesting can drastically increase IAV yields and possibly the yield of other viruses. To our knowledge, this is the first report to show the potential of this device for viral vaccine production.

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Section: Perfusion Bioreactor Cultivationssupporting

confidence: 60%

Section: Virus Titrationmentioning

confidence: 99%

Section: Virus Titrationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode

Coronel

Gränicher

Sandig³

et al. 2020

Front. Bioeng. Biotechnol.

Self Cite

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show abstract

“…Cell culture-based platform has been established as a viable alternative for the manufacturing of in uenza vaccines, particularly advantageous in case of a pandemic, due to its exibility, scalability and lower potential constraints of egg shortages (Harding and Heaton 2018; Milian and Kamen 2015). Particularly, the lately developed disposable equipment enables the fast and handy manufacturing of in uenza vaccines (Coronel et al 2019). Various continuous cell lines have been characterized for the propagation of in uenza viruses, such as HEK293, Vero, PER.C6, EB66, PBG.…”

Section: Introductionmentioning

confidence: 99%

Highly Efficient Production of an Influenza H9N2 Vaccine Using MDCK Suspension Cells

Jia

Lai³

et al. 2020

Preprint

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H9N2 subtype avian influenza virus poses a constant threat to the poultry industry and the control of the disease leans upon the use of effective vaccines. As an alternative to the conventional chicken embryonated eggs, animal cell culture could overcome the limitations of egg supplies and upgrade the manufacturing of avian influenza vaccines for poultry. Development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good growth and production performance is required. In this work, an adherent MDCK cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the influenza virus propagation in this MDCK cell line was evaluated and was improved with the medium exchange at time of infection as well as optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. Evaluation of MDCK cell growth and H9N2 virus propagation in the bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 14000 virions/cell. With the purified H9N2 virus harvested from the bioreactor, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.

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

Gränicher

Tapia

Behrendt

et al. 2020

Biotechnology Journal

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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

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Influenza A virus production in a single-use orbital shaken bioreactor with ATF or TFF perfusion systems

Cited by 30 publications

References 36 publications

Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode

Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode

Highly Efficient Production of an Influenza H9N2 Vaccine Using MDCK Suspension Cells

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

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