The Process Control And Optimization Of PCV2 Virus-Like Particles Vaccine Production Based On PAT

Improving the quality and reducing the cost of industrial products to get the most profits is the essential target of all the pharmaceutical manufacturers.Process analytical technology(PAT)requires the recognition of critical process parameters and the critical product quality as well as the interaction between them.The on-line monitoring of these critical parameters can ensure the real-time regulating of the bioprocess,thus realizing the finial purpose of product quality assurance.Porcine circovirus type 2(PCV2)is a dangerous pathogen for pigs,causing the post-weaning multi-systemic wasting syndrome(PMWS)and other fatal infectious diseases in pigs.At present,vaccination is the main protective measure.Virus-like particles(VLPs)subunit vaccine is the second generation of the vaccinal development,containing one or several viral capsid protein without the viral genome.VLPs can stimulate both innate and adaptive immune responses.VLPs are safe for vaccinal candidates because they are lack of the viral DNA.So VLPs are getting more and more attention because of their protective clinical effects and easily scale-up production methods.Based on PAT,this study explored the critical process parameters as well as the interaction among them,with the target of recognizing the underling mechanism of VLPs bioreactor production process of SF 9 cells.Besides,the identification of optimal feeding time,the infection time and harvest time were also major task in this study.Overall,the principal schedule was to establish an efficient production strategy.Firstly,based on multi-parameters analysis of the physiological parameters of the Sf 9 cells from the batch culture,this study found that the capacitance linearly correlated with the viable cell density,which indicated that the capacitance could server as the on-line signal of the viable cell density in the culture.It also found the linear relationship between specific growth of capacitance(μCap)and the growth rate of Sf 9 cell,so μCap could be used as on-line signal of the cell growth rate.On-line analysis of the CO2 volumetric fraction in the off-gas showed a linear relationship with the specific oxygen uptake rate of the culture,which could indicate the change of specific oxygen uptake rate of cell culture on-line.On-line specific oxygen up take rate and on-line critical frequency correlated with the cell metabolic activity state,which could be used as on-line indications of the metabolic state.Secondly,based the multi-parameters analysis mentioned above.This study identified the optimal feeding time using the critical frequency as on-line indicative signal,realized the high density Sf 9 cultivation on the bioreactor.This improved the viable cell density from 1.2× 106 cells/ml to 2.1 × 107 cells/ml,increased by 1.75 times.With the comparison of cell growth activity and the metabolic activity between the batch culture and the fed-batch culture,it found that the feeding strategy improved the viable cell density and the specific oxygen uptake rate as well as the specific glucose uptake rate greatly,prolonged the time in which the cell experienced these high rates.The feeding strategy enhanced the TCA cycle and improved the global metabolic state.Lastly,based on the physiological multi-parameters correlation analysis between batch culture and fed-batch culture,used the critical frequency as on-line feeding signal,the μCap as on-line infecting signal,the max capacitance as on-line vaccine harvest signal,this study performed the vaccine production successfully.The volumetric productivity of fed-batch culture was improved by 76%than the productivity in the batch culture.The volumetric productivity of the fed-batch culture was 150 mg/L,higher than the traditional production method(120 mg/L)by 25%.It could get the max productivity 24 hrs earlier than the traditional method,which reduced the production cycle and improved the utilization factor of equipment significantly.Besides,optimizing the production process based on the PAT could reduce the optimal time-cycle.The access of kinds of physiological parameters could facilitate the manufacturing process,assure the homogeneousness of the different production batch as well as the product quality.Based on PAT,this study developed a PCV2 vaccine production system by baculovirus insect cell expression system on the 5 L bioreactor.The system turned out to be an effective PCV2 production system.It could be recognized a breakthrough for the bioprocess study based on PAT.