Seed Train Intensification and TFDF-Based Perfusion for MDCK Cell-Based Influenza A Virus Production

Abstract

The production of influenza A virus (IAV) using Madin-Darby Canine Kidney (MDCK) cells is a key strategy for efficient influenza vaccine manufacturing. However, challenges remain in optimizing cell culture processes for higher yield and efficiency. This study aims to evaluate different process intensification strategies on two distinct clonal MDCK suspension cell lines (C59 and C113) for improved IAV production. A semi-perfusion strategy was used to push cells towards high cell density (HCD), achieving up to 17 × 10⁶ C113 cells/mL and 42 × 10⁶ C59 cells/mL, respectively. Next, a Tangential Flow Depth Filtration (TFDF)-based perfusion process with direct harvest during IAV production was established, resulting in high titers and a 10-fold higher space-time yield for C59 and a 4-fold improvement for C113 compared to batch operation. In addition, the suitability of N-1 perfusion was evaluated for batch and intensified fed-batch processes. Cells taken from the N-1 perfusion showed different cell-specific growth rates, but this had no effect on virus titers except for processes started from oxygen-deprived precultures. Finally, comparable virus titers were obtained when the production bioreactor was directly inoculated from an HCD cryovial. Taken together, seed train intensification and TFDF-based perfusion majorly reduced process times and improved IAV production.