Facing the global increasing demands for effective and safe vaccines for human and animal use, progressive process strategies are demanded in cell culture-based viral vaccine production to overcome the limitations of conventional batch cultures. Many factors in production process including cell concentrations at time of infection (TOI), metabolic status of cells at TOI, multiplicity of infection (MOI) at TOI and residence time affect the yield and quality of virus. Ideally, high cell density (HCD) process may achieve high virus production when using fed-batch or perfusion strategies, despite high cell concentrations may encounter so-called cell density effect, which is a reduction in the cell-specific virus yield. This work aims to develop a simple and effective feeding strategy in HCD process to obtain high and stable virus production in vaccine manufacturing. By cultivating a single-cell suspension cell line in an in-house serum-free medium, we analyzed the changes in nutritional needs of cells especially carbohydrates and amino acids, and by-product accumulations like ammonia and lactate in a HCD process between the infection group and mock group. We also detected the virus-induced apoptosis by flow cytometry to analyze how infection altered the original cell batch process. Based on the results of comprehensive and systemic analysis of nutrition supply and virus-induced apoptosis, several feeding strategies were designed for the virus production to be tested. By analyzing the final nutrition status, virus titer, virus infectivity, cell-specific virus yields and particle morphology, optimal results were achieved by performing a 1:1 dilution with fresh culture medium at the time of infection. Larger proportion of intact virus particles, fewer broken particles and fewer abnormal structures were observed. In addition, this strategy resulted in more infectious virions and higher HA titre (214 HAU/25μl) without reduction of cell-specific virus yields. Above all, a robust cell culture-based viral vaccine production process was established to provide a significant increase in productivity and efficiency that is promising to be applied into large-scale vaccine manufacturing.

Process optimization for high cell density cultivations in cell culture-based viral vaccine production

253rd National Meeting (2017)

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