| In recent years, with the rapid development of animal cell culture technology, it holds tremendous advantages for bio-pharmaceutical production in flexibility, efficiency and safety. Particularly, such cell culture-based technology is being applied in viral vaccine production and expected to replace the traditional processes. However, current animal cell culture-based processes, especially at high cell density, have no distinct advantage in virus propagation efficiency over traditional chicken embryo method, leading to being less cost-effective. Therefore, it becomes very urgent to understand the effects of culture conditions, such as cell physiological status, environmental nutritional supply and operating parameters, and viral infection parameters on cell growth, cell density maintaining, and viral infection and replication. It is anticipated that optimization in these parameters would result in drastic enhancement in virus production efficiency.In influenza vaccine production processes based on large-scale animal cell culture technology, the virus yield is primarily dictated by two parameters including cell growth and virus propagation efficiency. Hence, in this study, the effects of adhesive substrates (microcarriers) and growth stimulants (serum) on MDCK cell growth, as well as the effects of viral infection parameters on virus propagation were investigated at first to establish the initial culture process suitable for MDCK cell growth and influenza virus production. It is well known that there is a tight correlation between virus propagation and cellular physiological status. Considering that conditions such as environmental nutrients supply and operating parameters of bioreactor can affect cellular physiological status, the effects of these culture conditions on virus propagation were subsequently studied. Finally, optimization in nutritional compositions of maintaining medium and bioreactor-operating parameters was performed to develop a process for both high-density MDCK cell culture and high-efficient influenza vaccine production, which would meet the needs of future large-scale industrial production.Initially, it was found that both Cytodex-1 and Cytodex-3 microcarriers well supported growth of MDCK cells, and fetal bovine serum (FBS) was significantly better than newborn bovine serum (NBS) for cell growth. Specifically, MDCK cells grew well with a highest cell density of 5.5×106 cells/ml with 2 g/L Cytodex-1 or 3 g/L Cytodex-3, DMEM as the basal medium and 7.5%(v/v) FBS supplemented. Three critical parameters including TPCK-trypsin concentration, MOI and TOI for influenza virus infection and replication showed significant impacts on virus propagation, and it was suitable for virus propagation when TPCK-trypsin concentration, MOI and TOI were 5 mg/L,0.01 and 72 h, respectively. Based on these findings, an initial process suitable for MDCK cell growth and influenza virus production in bioreactor was established, and HA titer and specific virus yield (Svy) of influenza virus were 210.13±0.18 HA units/50 μl and (6.09±0.44)×103 virions/cell, respectively.Then, in order to improve influenza virus production efficiency based on such a process, the effects of key nutrient components in maintaining medium on cell density maintaining, cell metabolism and virus propagation during virus production phase were investigated. Then, the nutritional and metabolic requirements for influenza virus propagation in MDCK cells were analyzed, and maintaining medium was optimized. It was found that medium change during virus production phase could significantly improve Svy, indicating the importance of nutritional supply to improve virus propagation. It was also shown that during virus production phase, a slight increase in glucose concentration maintained a higher cell density, an increase in glutamine concentration in maintaining medium supported both better cell density maintaining and higher virus production efficiency, and an enhancement in concentrations of other amino acids promoted virus production efficiency. During virus production phase, supplementation of 0.75 mmol/L sodium butyrate (NaBu) could significantly improve influenza virus yield. It was possibly because a higher proportion of G0/G1 cells and lower percentage of apoptotic cells were present at the presence of NaBu, which could potentially improve virus infection and extend virus propagation time. Based on these results, a maintaining medium named Optimized-MM was developed and optimized and influenza virus HA titer, HA antigen protein concentration and Svy were improved by (159.37±31.82)%, (34.29±2.03)% and 166.17±37.50%, respectively.Further, bioreactor-operating parameters including dissolved oxygen, pH and temperature during both cell growth phase and virus production phase were optimized. The results indicated that within the set ranges, pH and temperature exerted significant impacts on influenza virus production efficiency. Specifically, virus production efficiency was significantly improved when pHpi was set at 7.2. Under this condition, a higher percentage of cells at G0/G1 phase and lower percentage of apoptosis cells were present, the expression of viral RNA polymerase was up-regulated and the inhibition of anti-virus protein to viral RNA polymerase activity was attenuated. All these collectively favored virus infection, viral RNA synthesis and intracellular viral propagation. Virus production efficiency was highest when the temperature of both cell growth phase and virus production phase were set at 35℃. Under this condition, a higher percentage of G0/G1 phase cells and lower percentage of apoptosis cells were observed. Additionally, the expression of viral RNA polymerase was promoted, the inhibitory effects of anti-virus cellular protein on viral RNA polymerase activity were reduced, and the positive effect of pro-virus cellular protein on viral RNP nuclear export were enhanced, which were beneficial to virus infection, viral RNA synthesis and viral RNP nuclear export, and resulted in effective intracellular viral propagation.Finally, according to the studies above, environmental nutrients supply and operating parameters of bioreactor were determined for high-efficient production of influenza virus, and a optimized process was established based on the initial process above thereof. There was no significant difference in MDCK cell growth between the initial process above and the optimized process here during both cell growth phase and density maintaining phase (i.e., virus production phase). However, influenza virus HA titer, HA antigen protein concentration and Svy of the optimized process reached 213.00 HA units/50 μl,16.77±0.37 μg/ml and (41.31±0.98)×103 virions/cell, which were 7.36±0.90,1.89±0.90 and 6.83±1.02 times that of the initial process, respectively. Furthermore, a better stability of the virus was also achieved in the optimized process.Taken together, an efficient process for influenza vaccine production based on the large-scale MDCK cell culture was developed, which lays a foundation for industrial production of influenza vaccine. Moreover, ta comprehensive understanding of the impacts of essential culture parameters on influenza virus propagation in cell culture ensures the rational development and optimization of viral vaccine production processes. |
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