Influenza has been a threat to human society as an epidemic. Its regional explosion as emergency requires hundreds of million doses of vaccine within a short period. Embryonated chicken eggs were conventionally used in influenza vaccine manufacturing, which owns inherent limitations. Cell culture based influenza vaccine production processes were emerging as an flow of the tide to substitute embryonated chicken eggs in the past decade. Madin-Darby canine kidney (MDCK) cells are moving up to the front as a favorite host cell line for propagating influenza virus considering its superiority of robust growth and high susceptibility to influenza virus. However, MDCK cells are prone to adhere in serum-containing medium, or form clumps in suspension culture. Lacking of robust serum-free medium and proper process, state of art of the cell-based influenza vaccine production processes are based on either serum-containing medium or costly microcarriers which hurdles it to become dominant. This work aims to develop a better serum-free influenza virus production process with high efficiency and scalability. An in-house serum-free medium suitable for suspension culture of MDCK cells was developed by rational design and further optimization. We evaluated the growth of MDCK cells in the serum-free medium formulated with the key components at optimal concentrations, which were designed according to its dynamic rates and stoichiometry ratios. It supports the peak viable cell density of 2.0×107 cells/ml and the specific growth rate of &gt;0.9 d−1. Considering the mass and energy metabolic requirements, we established the virus amplification process by nutrient concentration optimization based on consumption rates. Glucose and fast metabolized amino acids were supplemented at the time of infection. By setting optimized MOI (multiplicity of infection) and TOI (time of infection), and supplementing proper amount of TPCK-trypsin based on tryptic activity, the viral titer of 212 (HAU/25 μl) was achieved in shake flask. The bench scale process was further scaled up to 6,000 L successfully. The final titer reached (211-212 (HAU/25 μl)) with strong immunogenicity. Eliminating serum and microcarriers, the work enables MDCK cells cultured in single-cell suspension without severe aggregation. The high-efficient large scale process for influenza virus production provides a potential alternative for upgrading of current influenza vaccine manufacturing.

Establishing a high-efficient serum-free process for influenza vaccine production using MDCK cells

253rd National Meeting (2017)

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