| Facing with the outbreak of influenza virus, animal cell culture technology was widely applied in influenza vaccine production due to its advantage over traditional embryonated egg-based production technology. How to quickly and efficiently develop robust and high quality cell-culture based influenza vaccine process had become the focus of research. Agitation in bioreactor, known as an essential operation parameter, played important roles, for example:not only to enhance fluid mixing; to accelerate mass transfer rates, but also to bring shear stress and microcarriers collision. Thus, choosing optimal agitation speed to balance mass transfer and shear stress was vital in vaccine process development. In this paper, cell growth, cell metabolism and virus replication were investigated both in microcarrier-based and single cell suspension culture.Firstly, we investigated the effects of agitation speed on MDCK cell growth, metabolism and virus replication in microcarrier-based cell culture. The results showed that:during cell growth phase, with agitation speed increased, cell attachment time, glucose uptake rate and ammonium, lactate release rate increased, while cell attachment rate constant and maximum cell density decreased. The conversion coefficient of lactic acid to glucose (Ylac/gluc) showed in a different manner, with mild agitation speed range (30～90 rpm), the Ylac/gluc decreases with increasing agitation, which implied a more sufficient glucose utility; under excess agitation (120,150 rpm), the Ylac/gluc was kept at a higher level. During virus replication phase, no differences were observed both in cell growth performance and virus titer. Apparent cell death rate and LDH activity increased with increasing agitation speed. Virus titer were 5.0,6.0,6.0 log2(HAU/50 μl) at 30,90 and 150 rpm, and the maximum Svy value were (439.04±5.06), (817.61±19.29), (914.18±17.58)virions/cell.Secondly, the effects of agitation speed on cell growth, metabolism and virus replication were also performed in single cell suspension culture. The following results were obtained: During cell growth phase, cell density, glucose uptake rate and ammonium, lactate release rate increased with agtition intensity enhansed, while the Ylac/gluc decreased. Keeping the same speed all the culture process, virus titer were 6.0, (6.8±0.4), (6.7±0.5) log2(HAU/50 μl) at 30, 90 and 150 rpm, and the maximum Svy value were (439.04±5.06), (817.61±19.29), (914.18±17.58) virions/cell. Descending agitation speed after high cell desity led to fast cell death, and no virus titer was measured at 30 and 90 rpm. Under overlay aeration condition, cell death rate of 90 rpm decreased, while the death rate at 30 rpm showed no significant changes. Meanwhile, no virions were detected at 30 rpm, while virus titer were 9.0 log2(HAU/50 μl) at 90 rpm. Keeping the same speed all the culture process with overly aeration, virus titer were 8.0,10.0,12.0 log2(HAU/50μl) at 30,90 and 150 rpm, and the maximum Svy value were (3082.00±213.04), (5535.88±189.70), (13234.41±908.48) virions/cell. The effects of dissolved oxygen (DO) on virus replication were also deterimined in 3 L bioreactor. The results showed that cell density showed no significant differernces. virus titer were 11.0,13.0 log2(HAU/50μl) at DO10%, DO50%, the maximum Svy value were (6192.23±193.65), (26753.49±507.27) virions/cell, respectively.In this paper, we studied the different roles of agitation speed on cell growth, metabolism and virus replication. The results had deepened our outstanding of agitation, thus, providing guidance for further development of industrial-scale vaccine production processes. |
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