A new porcine suspension cell line (PBG.PK-21) provides efficient production for influenza and yellow fever vaccine viruses
June 17-22, 2018
Shifting from egg-based influenza and yellow fever vaccine production towards efficient cell culture-based manufacturing is one of the biggest challenges in viral vaccine manufacturing. Innovative bioprocesses, such as high cell density  or continuous virus cultivations  improving cell-based viral vaccine production have been shown over the past years. However, finding the right high-yield cell substrate is still pending for many important viruses. Ideally, such host cell lines should enable the efficient production of several virus strains to set-up manufacturing platforms.
Here, we report on a novel suspension cell line (PBG.PK-21 (ProBioGen) derived from immortal porcine kidney cells. These cells were first adapted to chemically defined virus production medium CD-U5 (ProBioGen) and agitated suspension culture. Despite its robust growth these cells were not yet suitable for virus manufacture due to a chronic infection with porcine circovirus 1 (PCV1) that is often found in porcine cell lines. The cells were cured from infection after siRNA mediated suppression of the contaminating virus followed by single cell cloning. Clone number 21 proven to be free from PCV1 over multiple passages and distinguished by high peak cell densities and good productivity for other viruses was chosen for further analysis.
Cell growth, cell metabolism and virus production were characterized in shake flasks and bioreactors. Cell concentration up to 8 x 106 cells/mL and a doubling time of 33 h were obtained in batch mode in CD-U5 medium. Furthermore, process intensification using either semi-perfusion in shake flasks or hollow fiber-based perfusion with an ATF2 system coupled to a 0.6 L wv stirred tank bioreactor system was evaluated. In this system, cell densities up to 42 x 106 cells/mL were achieved with a cell-specific perfusion rate of 0.07 nL/cell/day.
After optimization of influenza A/PR/8/34 (H1N1) virus production, HA titres of 3.3 log(HAU/100µL) with a cell-specific yield of 5200 virions/cells were achieved in bioreactor fed-batch mode. The PBG.PK-21 suspension cell line also shows potential to be used in a modern cell-based swine influenza vaccine production process for the veterinary market. Indeed, similar competitive virus titres (3.4 log(HAU/100µL)) and cell-specific yields were found for the swine influenza A/Bakum/1832/00 (H1N2) virus in batch mode. Finally, tests were also conducted regarding yellow fever virus production (live-attenuated 17DD YFV, RKI). In first scouting experiments, promising titres up to 3 x 106 PFU/mL were obtained in batch mode.
First attempts towards process intensification using ATF-based perfusion systems with manually-adapted perfusion rate showed successful maintenance of cell-specific influenza A virus yields at high cell density. Titres up to 4 log(HAU/100µL) were obtained with 42 x 106 cells/mL. The glycosylation profile of influenza A/PR8/34 produced in PBG.PK-21 cell line was also analysed and will be compared to profiles of other cell lines.
Overall, PBG.PK-21 suspension cells show a great potential to become the cell substrate of choice for many existing and new viral vaccine processes in next generation high-yield human as well as veterinary vaccine manufacturing.
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Gwendal Gränicher, Yvonne Genzel, Udo Reichl, Katrin John, Volker Sandig, and Alexander Karlas, "A new porcine suspension cell line (PBG.PK-21) provides efficient production for influenza and yellow fever vaccine viruses" in "Vaccine Technology VII", Amine Kamen, McGill University Tarit Mukhopadhyay, University College London Nathalie Garcon, Bioaster Charles Lutsch, Sanofi Pasteur Eds, ECI Symposium Series, (2018). http://dc.engconfintl.org/vt_vii/48