Quality By Design Driven Process Development Of Mammalian Cell Culture For The Production Of Vaccine

As one of the important biological products,vaccine is a key means for humans to prevent epidemics.Due to the complexity of the vaccine production process,product quality is hard to control.In recent years,international drug regulatory agencies have imposed stricter controls on the development of biomedical process engineering and product quality.The core is to drive the development of biological processes based on the principle of“Quality by Design?QbD?”.In this study,the producing process of severe fever with thrombocytopenia syndrome?SFTS?vaccine by Vero cells and foot-and-mouth disease vaccine by BHK-21 cell were studied by QbD principle.First,serum-free media of Vero cells were selected by screening commercial serum-free media and applied to a microcarrier suspension culture system.Guided by QbD,the fermentation process of Vero cells to produce the SFTS vaccine was optimized and a suitable design space and operation space were found.The 3000 L scale producing the foot-and-mouth disease vaccine was successfully scaled-down to 10 L scale,and QbD driven process research was then carried out in this scale-down model.The main research conclusions are divided into four parts.?1?Based on the basic process of Vero cell microcarrier suspension culture,the culture time was shortened from 7 days to 4 days by adding non-essential amino acids?NEAA?,changing the rotation speed and increasing the amount of liquid exchange.By screening commercial serum-free media,serum-free medium OptiPRO SFM of Vero cells was selected,and 0.2%NBS was added as a low serum culture protocol.It was proved that the method can be applied to a microcarrier suspension culture system.By adding the feed to the low serum medium,the conversion from the liquid exchange culture to the fed-batch culture was achieved,and the maximum cell density was 25%higher than average level.?2?Sixteen single factor experiments were carried out on the virus culture stage of SFTS vaccine production,and seven potential key process parameters?MOI,CB4,CB5,H₂O₂,ethanolamine DMSO and HSA?were selected.The Plackett-Burman method was applied as a factor screening experiment,through which CB4 and H₂O₂ were found significantly promoted the virus yield.Ethanolamine significantly increased the cell density at harvest.Based on this result,we selected CB4,H₂O₂ and ethanolamine as critical process parameters?CPPs?for the following response surface method experiment.Through the RSM experiment,a mathematical model of the relationship between CPPs and critical quality attributes?CQAs?was established,and 5000 tests of monte carlo simulation experiments were carried out to find the design space and operation space of the producing process.Finally,we scaled-up the production scale to a 2 L bioreactor,demonstrating the feasibility of this process in a STR-type bioreactor.?3?We established a scale-down model based on the production scale of 3000 L foot-and-mouth disease vaccine.Firstly,through the calculation and comparison of various hydrodynamic parameters in different scale reactors,the constant P/V is finally determined as the scale-down strategy of production scale from three scale-down strategies.The computational fluid dynamics?CFD?simulation was used to verify the scale-down strategy.The simulation results were basically consistent with the calculation results,indicating the feasibility of the scale-down model.Compared with the actual culture,there was no significant difference in cell culture and product quality in different scale reactors,which proved the reliability of the model scale-down strategy in this study.It was proved that the scale-down strategy followed the regular pattern of bioreactor scale-down process,and a technical guiding principle of the mammalian cell culture process scale-down model is successfully established.?4?Based on the established scale-down model,we apply the QbD principle to the development of the process for foot-and-mouth disease.First,three CQAs were identified.Risk assessment of CQAs was performed by the failure mode and effect analysis?FMEA?method.The mathematical model of CPPs and CQAs was established by using response surface method,and four CPPs were determined.The monte carlo simulation experiment was used to determine the process design space and operation space of the vaccine production.The design space and the operation space were verified by simulation and actual production.The overall defect rate was significantly reduced from 18.33%to 1.67%,which was consistent with our expectations.This result proves the validity of the design space and the operation space,and illustrates the correctness of the application of the QbD principle methodology.Finally,we established a batch model by collecting the process parameters of 100batches that can be used to guide subsequent vaccine production.