Introduction: The spread of viral diseases, especially newly emerging infectious diseases, is a serious threat to humans and the constancy of the world. The best way to control epidemic diseases is to make the society more accessible to the vaccination program. The speed of the spread of emerging infectious diseases has recently created new challenges for vaccine manufacturers, and it is also challenging to produce vaccines on a large scale. Therefore, new vaccine platform technologies may shorten this cycle and accelerate vaccine development. In the process of producing vaccines based on viral vectors, the main step is the production of host cells with appropriate cell density. Increasing the cell density for vector production helps to improve volumetric production while maintaining maximum specific production. The choice of cultivation mode is crucial because of its effect on the way of providing nutrients and removing metabolites in cell concentration, product titer and volume adenovirus vector production that can be achieved. We set up efficient adenovirus rAd26 production in animal component-free conditions employing a well-characterized HEK 293 cell line cultivated in the flasks and bioreactor, a scalable system for cell culture that can be used to study process development and optimization for vaccine production. HEK 293 it acts as a host for the development of recombinant adenoviral vectors. The results obtained from 200L SUB demonstrated that the virus concentration peak occurred consistently 72 h after infection and the virus yields were strongly corresponding to the process parameters, such as cell density at infection & MOI.
Methods: In the present study, the HEK 293 cells were infected with recombinant adenovirus serotype 26 (rAd26), and the effects of critical process parameters (CPPs) with 1.4×106 cells/mL with 95% viability and including the multiplicity of infection (MOI) = 3, 6, 9, 12, and 15 and were the specific productivity was measured at 120 hpi in various SF2Ls investigated experimentally. The results of small-scale experiments in 2 L shake flasks (SF 2L) demonstrated that MOI could affect the cell proliferation and viability.
Results: The results at these experiments showed that VCD = 1.4 × 10-6 cells/mL and MOI = 9 yielded TCID50 /mL = 108.9, at 72 h post infection (hpi), while the virus titer at MOI = 3 and 6 was lower compared to that of MOI=9 and on TCID50 /mL and MOI = 3 and 6 were less efficient. Moreover, our findings showed that MOIs > 12 did not have a positive effect, 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 yielding high-titer rAd26 manufacturing, TCID50/mL = 108.9, at 72 hpi.
Conclusion: adenovirus rAd26-S-CoV-2 synthesis at MOIs 3 and 6 IU/ml showed that the viruses were unable to infect cells quickly (more than 120 hours). Premature cell death and lysis resulted in a considerable decrease in cell density and viability, and the effectiveness of infection as a result of the consequent titer was not proportionate to the rise in the MOI value. When virus particles infect cells by the batch process, cell development stops 24 to 48 hpi, they start to collect in the cells 48 to 72 hours later, reach their peak titer, and then the cells die. The viral multiplication factor, or MOI, of the process, was discovered to be associated with cell density and might range from 108 to 1010.Produce high titers virus requires an intricate understanding of the critical process parameters to ensure that high titers can be achieved in a reproducible manner.