Sars-cov-2 production in a scalable high cell density bioreactor

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has demonstrated the value of pursuing different vaccine strategies. Vaccines based on whole viruses, a widely used vaccine technology, depend on efficient virus production. This study aimed to establish SARSCoV-2 production in the scalable packed-bed CelCradle™ 500-AP bioreactor. CelCradle™ 500-AP bottles with 0.5 L working volume and 5.5 g BioNOC™ II carriers were seeded with 1.5 × 10⁸ Vero (WHO) cells, approved for vaccine production, in animal component-free medium and infected at a multiplicity of infection of 0.006 at a total cell number of 2.2–2.5 × 10⁹ cells/bottle seven days post cell seeding. Among several tested conditions, two harvests per day and a virus production temperature of 33^◦C resulted in the highest virus yield with a peak SARS-CoV-2 infectivity titer of 7.3 log₁₀ 50% tissue culture infectious dose (TCID₅₀)/mL at 72 h post-infection. Six harvests had titers of ≥6.5 log₁₀ TCID₅₀/mL, and a total of 10.5 log₁₀ TCID₅₀ were produced in ~5 L. While trypsin was reported to enhance virus spread in cell culture, addition of 0.5% recombinant trypsin after infection did not improve virus yields. Overall, we demonstrated successful animal component-free production of SARS-CoV-2 in well-characterized Vero (WHO) cells in a scalable packed-bed bioreactor.