Influenza viruses pose a significant threat to global health, with the potential to trigger pandemics. Vaccination remains the primary strategy to reduce illness and death during such outbreaks. Addressing the urgent need for readily available vaccines, a new study introduces an advanced manufacturing process for inactivated influenza H5N1 vaccines. This innovative process leverages Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system, offering significant improvements in vaccine production.

The study focused on transitioning MDCK cells from serum-containing (SC) medium to various commercial SF media. The selected SF medium, Plus-MDCK, was tested in different bioreactor systems to assess scalability. The results demonstrated no significant differences in cell growth across bioreactor sizes. In 7.5 L bioreactors, cell concentration reached 2.3×10^6 cells/mL within five days. After three days of infection, the maximum virus titers achieved were 1024 Hemagglutinin (HA) units/50 µL and 7.1±0.3×10^8 plaque-forming units (pfu)/mL.

Additionally, the concentration of HA antigen, determined by Single Radial Immunodiffusion (SRID), was 14.1 µg/mL, surpassing levels from SC medium. A mouse immunogenicity study indicated that the formalin-inactivated SF vaccine candidate, formulated with an alum adjuvant, could induce protective virus neutralization titers comparable to those from SC medium. H5N1 viruses produced in both SC and SF media showed identical antigenic reactivity with NIBRG14 standard antisera.

The pandemic potential of the highly pathogenic avian influenza H5N1 virus underscores the urgent need for an effective vaccine. Traditional egg-based vaccine production methods may not meet pandemic demands due to reliance on a steady egg supply. The cell-based approach, using cell lines such as MDCK, has gained significant interest for influenza virus production due to their ability to propagate various strains and produce high virus yields with substantial HA titers.

Transitioning to SF medium addresses several disadvantages of SC medium, including the risk of hypersensitivity reactions, batch variability, and potential contaminant introduction. SF medium reduces contamination risks and simplifies downstream purification steps, resulting in high-quality and consistently safe products. However, developing a viable SF medium process for both cell growth and virus replication remains challenging, requiring extensive empirical experimentation.

In dynamic environments like bioreactor cultivation, shear forces can significantly affect cell viability. Adding shear protectants like Pluronic F-68 and using bubble-free aeration methods help mitigate these effects. The study demonstrated that MDCK cells grown in Plus-MDCK medium with microcarriers in a perfusion bioreactor system achieved high virus titers (∼10^8 pfu/mL) without extensive adaptation procedures.

Interestingly, the cell density in SF medium was lower, but the yield of H5N1 antigen was higher compared to SC medium. This phenomenon may be due to differences in medium composition affecting cellular metabolism or protease activity involved in virus replication.

This study highlights the advantages of using SF medium in the cell-based manufacturing process for influenza vaccines. The Plus-MDCK medium supported both MDCK cell growth and H5N1 virus replication, resulting in a safe and efficient production process with fewer contaminants and high yields. Additionally, the cost of Plus-MDCK medium was found to be 55% lower than SC medium.

This innovative approach marks a significant step forward in ensuring efficient and reliable vaccine production, supporting global health preparedness against influenza pandemics.

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