Rethinking process development for influenza vaccine production

The propagation of influenza viruses in embryonated chicken eggs has been a common practice for vaccine manufacturing since the 1940's. In recent years, researchers from industry and academia have moved away from this traditional method towards cell culture-based production systems.

Our group contributes with a fully integrated influenza A virus (IAV) production process using suspension MDCK cells that are cultivated in chemically defined media. This process established could produce 300 vaccine doses per liter within 4–5 days and thus compete with egg-based and other cell-based production systems (Bissinger et al., 2021; Wu et al., 2021).

Nevertheless, the full potential of MDCK cells may not be exploited yet, and we thus move further by rethinking process development and by implementing state-of-the-art technologies. A single-cell cloning approach is applied to challenge current host cell options and to investigate whether monoclonal suspension MDCK cells can compete with the heterogonous cell cultures traditionally used in large-scale manufacturing. Clonal selection can result into a high number of candidate cell lines requiring detailed evaluation of growth performance and virus production capacities. We supported this evaluation by using a modern single-use microbioreactor system (ambr^®15) with an initial screening, followed by process optimization using a design of experiments (DoE) approach at 10–15 mL scale. This is feasible as this system allows for 24 parallel cultivations performed under tight process control, which results in fast, reliable outcomes under minimal medium use. After top clone selection, process development is finalized by scaling into disposable benchtop stirred tank bioreactors at a 2 L scale for validation (Zinnecker at al., 2023).

Figure 1: Generation of monoclonal MDCK suspension cell cultures using a CellCelector™. Thousands of MDCK cells were seeded into a nanowell-plate. Cells were efficiently separated from each other, yet co-cultured in the same medium. This allows all cells to contribute to the growth of the clonal cell lines, while maintaining their monoclonality. The integrated imaging system scanned automatically the nanowells on day 0 to identify the wells containing only one cell. After four days of monitoring, cells were automatically transferred for further expansion steps. — **Figure 1: Generation of monoclonal MDCK suspension cell cultures using a CellCelector™.** Thousands of MDCK cells were seeded into a nanowell-plate. Cells were efficiently separated from each other, yet co-cultured in the same medium. This allows all cells to contribute to the growth of the clonal cell lines, while maintaining their monoclonality. The integrated imaging system scanned automatically the nanowells on day 0 to identify the wells containing only one cell. After four days of monitoring, cells were automatically transferred for further expansion steps.

**Figure 1: Generation of monoclonal MDCK suspension cell cultures using a CellCelector™.** Thousands of MDCK cells were seeded into a nanowell-plate. Cells were efficiently separated from each other, yet co-cultured in the same medium. This allows all cells to contribute to the growth of the clonal cell lines, while maintaining their monoclonality. The integrated imaging system scanned automatically the nanowells on day 0 to identify the wells containing only one cell. After four days of monitoring, cells were automatically transferred for further expansion steps.

**Figure 2: Single-use bioreactor vessels at 2 L vs. 15 mL scale.**

© Nils Thomas

**Figure 2: Single-use bioreactor vessels at 2 L vs. 15 mL scale.**

© Nils Thomas

Bissinger, T.; Wu, Y.; Marichal-Gallardo, P.; Riedel, D.; Liu, X.; Genzel, Y.; Tan, W.; Reichl, U.: Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells. Biotechnology and Bioengineering 118 (10), pp. 3996 - 4013 (2021)

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publisher-version

Wu, Y.; Bissinger, T.; Genzel, Y.; Liu, X.; Reichl, U.; Tan, W.-S.: High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells. Applied Microbiology and Biotechnology 105 (4), pp. 1421 - 1434 (2021)

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Zinnecker et al. In preparation (2023)