High‐Titer Hepatitis C Virus Production in a Scalable Single‐Use High Cell Density Bioreactor

Hepatitis C virus (HCV) infections pose a major public health burden due to high chronicity rates and associated morbidity and mortality. A vaccine protecting against chronic infection is not available but would be important for global control of HCV infections. In this study, cell culture-based HCV production was established in a packed-bed bioreactor (CelCradle™) aiming to further the development of an inactivated whole virus vaccine and to facilitate virological and immunological studies requiring large quantities of virus particles. HCV was produced in human hepatoma-derived Huh7.5 cells maintained in serum-free medium on days of virus harvesting. Highest virus yields were obtained when the culture was maintained with two medium exchanges per day. However, increasing the total number of cells in the culture vessel negatively impacted infectivity titers. Peak infectivity titers of up to 7.2 log(10) focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 × 10(10) FFU, and a cell specific virus yield of up to 41 FFU/cell were obtained from one CelCradle™. CelCradle™-derived and T flask-derived virus had similar characteristics regarding neutralization sensitivity and buoyant density. This packed-bed tide-motion system is available with larger vessels and may thus be a promising platform for large-scale HCV production

SARS-COV-2 production in a single-use scalable high cell density bioreactor

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and the fast development of first-generation vaccines have demonstrated the value of applying a variety of vaccine technology platforms (1). Inactivated vaccines represent a well-known approach, and their manufacturing depends on high-yield virus production in appropriate biosafety level facilities. This study (2) aimed to establish efficient Vero (WHO) cell-based and animal component-free SARS-CoV-2 production in the CelCradle bioreactor (Esco Aster Pte Ltd.). The single-use culture vessels pre-packed with 0.1 L BioNOCII carriers are highly useful for small scale cultivation of adherent cell lines. Cultures were seeded with 1.5 × 10⁸ cells and total cell numbers peaked at 9 days post cell seeding (dpcs) with 2.7–2.8 × 10⁹ cells/vessel in non-infected cultures. To produce virus, cultures were infected at 7 dpcs at total cell numbers of 2.2–2.5 × 10⁹ cells/vessel at a multiplicity of infection of 0.006. Harvest of virus-containing supernatant twice instead of once per day improved the virus yield by 2–7 fold. Introducing a temperature shift from 37°C to 33°C upon the time of infection improved virus yield by 2–9 fold with a considerable decline of infectious titer only after 72 h post infection (hpi). Infectious titers peaked at 7.3 log₁₀ 50% tissue culture infectious dose (TCID₅₀)/mL at 72 hpi, and a total of 10.5 log₁₀ TCID₅₀ were produced in ~5 L (11 harvests). While trypsin has been reported to enhance SARS-CoV-2 spread in cell culture, addition of 0.5% recombinant trypsin from the time of infection did not affect virus yield. Overall, animal component-free production of SARS-CoV-2 in Vero (WHO) cells was successfully established in a single-use packed-bed bioreactor. β-propiolactone inactivated SARS-CoV-2 from this study was immunogenic and induced neutralizing antibodies in mice with mean 50% neutralization titers of 1/150 or 1/580 after three immunizations with doses containing 0.1 µg or 0.5 µg S1 protein, respectively. The CelCradle represents a scalable technology and is a small version of the TideXCell system applying single-use culture vessels with packed-bed volumes of up to 100 L. The potential rapid response to outbreaks with inactivated vaccines has been demonstrated in the SARS-CoV-2 pandemic (1). Vero cells are susceptible to a wide range of viral pathogens (3), and these scalable single-use bioreactors provide a high level of flexibility and potentially decreased response time for production of future emerging viruses for vaccine purposes. References: (1) Poland 2020 Lancet [PMID: 33065034], (2) Offersgaard 2021 Vaccines [PMID: 34209694], (3) Barret 2009 Expert Rev. Vaccines [PMID: 19397417