The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 virus) pandemic has shown the importance of pursuing various vaccine manufacturing strategies. In the present study, the HEK 293 cells were infected with recombinant adenovirus serotype 26 (rAd26), and the effects of critical process parameters (CPPs) including viable cell density (VCD) at infection time (0.5 x 10(6), 0.8 x 10(6), 1.4 x 10(6), 1.8 x 10(6), and 2.5 x 10(6) cells/mL), the multiplicity of infection (MOI) = 3, 6, 9, 12, and 15, and two aeration strategies (high-speed agitation with a sparging system and low-speed agitation with an overlay system) were investigated experimentally. The results of small-scale experiments in 2 L shake flasks (SF 2L) demonstrated that the initial VCD and MOI could affect the cell proliferation and viability. The results at these experiments showed that VCD = 1.4 x 10(6) cells/mL and MOI = 9 yielded TCID50 /mL = 10(8.9), at 72 h post-infection (hpi), while the virus titer at VCD = 0.5 x 10(6) and 0.8 x 10(6) cells/mL was lower compared to that of VCD = 1.4 x 10(6) cells/mL. Moreover, our findings showed that VCDs > 1.8 x 10(6) cells/m with MOI = 9 did not have a positive effect on TCID50 /mL and MOI = 3 and 6 were less efficient, whereas MOI > 12 decreased the viability drastically. In the next step, the optimized CPPs in a small scale were exploited in a 200 L single-use bioreactor (SUB), with good manufacturing practice (GMP) conditions, at RPM = 25 with an overlay system, yielding high-titer rAd26 manufacturing, i.e., TCID50/mL = 10(8.9), at 72 hpi.
