Background: Many details in cell culture-derived influenza vaccine production are still poorly understood and approaches for process optimization mainly remain empirical. More insights on mammalian cell metabolism after a viral infection could give hints on limitations and cell-specific virus production capacities. A detailed metabolic characterization of an influenza infected adherent cell line (MDCK) was carried out based on extracellular and intracellular measurements of metabolite concentrations. Results: For most metabolites the comparison of infected (human influenza A/PR/8/34) and mock-infected cells showed a very similar behavior during the first 10-12 h post infection (pi). Significant changes were observed after about 12 h pi: (1) uptake of extracellular glucose and lactate release into the cell culture supernatant were clearly increased in infected cells compared to mock-infected cells. At the same time (12 h pi) intracellular metabolite concentrations of the upper part of glycolysis were significantly increased. On the contrary, nucleoside triphosphate concentrations of infected cells dropped clearly after 12 h pi. This behaviour was observed for two different human influenza A/PR/8/34 strains at slightly different time points. Conclusions: Comparing these results with literature values for the time course of infection with same influenza strains, underline the hypothesis that influenza infection only represents a minor additional burden for host cell metabolism. The metabolic changes observed after12 h pi are most probably caused by the onset of apoptosis in infected cells. The comparison of experimental data from two variants of the A/PR/8/34 virus strain (RKI versus NIBSC) with different productivities and infection dynamics showed comparable metabolic patterns but a clearly different timely behavior. Thus, infection dynamics are obviously reflected in host cell metabolism.

With an increasing demand for efficacious, safe, and affordable vaccines for human and animal use, process intensification in cell culture-based viral vaccine production demands advanced process strategies to overcome the limitations of conventional batch cultivations. However, the use of fed-batch, perfusion, or continuous modes to drive processes at high cell density (HCD) and overextended operating times has so far been little explored in large-scale viral vaccine manufacturing. Also, possible reductions in cell-specific virus yields for HCD cultivations have been reported frequently. Taking into account that vaccine production is one of the most heavily regulated industries in the pharmaceutical sector with tough margins to meet, it is understandable that process intensification is being considered by both academia and industry as a next step toward more efficient viral vaccine production processes only recently. Compared to conventional batch processes, fed-batch and perfusion strategies could result in ten to a hundred times higher product yields. Both cultivation strategies can be implemented to achieve cell concentrations exceeding 10⁷ cells/mL or even 10⁸ cells/mL, while keeping low levels of metabolites that potentially inhibit cell growth and virus replication. The trend towards HCD processes is supported by development of GMP-compliant cultivation platforms, i.e., acoustic settlers, hollow fiber bioreactors, and hollow fiber-based perfusion systems including tangential flow filtration (TFF) or alternating tangential flow (ATF) technologies. In this review, these process modes are discussed in detail and compared with conventional batch processes based on productivity indicators such as space-time yield, cell concentration, and product titers. In addition, options for the production of viral vaccines in continuous multi-stage bioreactors such as two- and three-stage systems are addressed. While such systems have shown similar virus titers compared to batch cultivations, keeping high yields for extended production times is still a challenge. Overall, we demonstrate that process intensification of cell culture-based viral vaccine production can be realized by the consequent application of fed-batch, perfusion, and continuous systems with a significant increase in productivity. The potential for even further improvements is high, considering recent developments in establishment of new (designer) cell lines, better characterization of host cell metabolism, advances in media design, and the use of mathematical models as a tool for process optimization and control.

(2009). Continuous cell lines as a production system for influenza vaccines. Expert Review of Vaccines: Vol. 8, No. 12, pp. 1681-1692. doi: 10.1586/erv.09.128

Influenza viruses are a major public health burden during seasonal epidemics and a continuous threat due to their potential to cause pandemics. Annual vaccination provides the best protection against the contagious respiratory illness caused by influenza viruses. However, the current production capacities for influenza vaccines are insufficient to meet the increasing demands. We explored the possibility to establish a continuous production process for influenza viruses using the duck-derived suspension cell line AGE1.CR. A two-stage bioreactor setup was designed in which cells were cultivated in a first stirred tank reactor where an almost constant cell concentration was maintained. Cells were then constantly fed to a second bioreactor where virus infection and replication took place. Using this two-stage reactor system, it was possible to continuously produce influenza viruses. Surprisingly, virus titers showed a periodic increase and decrease during the run-time of 17 days. These titer fluctuations were caused by the presence of defective interfering particles (DIPs), which we detected by PCR. Mathematical modeling confirmed this observation showing that constant virus titers can only emerge in the absence of DIPs. Even with very low amounts of DIPs in the seed virus and very low rates for de novo DIP generation, defective viruses rapidly accumulate and, therefore, represent a serious challenge for continuous vaccine production. Yet, the continuous replication of influenza virus using a two-stage bioreactor setup is a novel tool to study aspects of viral evolution and the impact of DIPs.