Rapid process development of serum-free pseudorabies virus production with the Quality by Design approach.

This study described a successful application of the Quality by Design (QbD) approach to pseudorabies virus (PRV) production process development in a fixed-bed bioreactor using the serum-free medium (SFM). The innovated tube-fixed-bed bioreactor was used as a scale-down model of the fixed-bed bioreactor for process development. Risk analysis was performed using Ishikawa diagram combined with failure mode effects analysis (FMEA). The comparative experiment was performed to screen proper medium for adherent African green monkey kidney (Vero) cells from three commercially available SFMs (VP-SFM, ProVERO-1 and Vero-A). The Vero-A medium showed as an outstanding one for further study. The PRV titer in harvest medium was consider as Critical Quality Attribute (CQA) and the Critical Process Parameters (CPPs) [time of infection (TOI), multiplicity of infection (MOI) and initial inoculation cell density] ranked high with risk priority number (RPN) were taken into design of experiment (DoE) methodology. Then prediction model of PRV production process was established and a robust PRV production process was explored. Under the robust setpoint conditions, the Xcell 1 L laboratory-scale fixed-bed bioreactor yielded PRV titer up to 7.87 log10 TCID50/mL at 3 dpi, which was comparable with that in the tube-fixed-bed bioreactor. Combination of the tube-fixed-bed bioreactor and QbD approach could further accelerate the development of a robust virus production process.