The baby hamster kidney-21 (BHK-21) cell line is a continuous cell line used to propagate foot-and-mouth disease (FMD) virus for vaccine manufacturing. BHK-21 cells are anchorage-dependent, although suspension cultures would enable rapid growth in bioreactors, large-scale virus propagation, and cost-effective vaccine production with serum-free medium. Here, we report the successful adaptation of adherent BHK-21 cells to growth in suspension to a viable cell density of 7.65 × 10⁶ cells/mL on day 3 in serum-free culture medium. The suspension-adapted BHK-21 cells showed lower adhesion to five types of extracellular matrix proteins than adherent BHK-21 cells, which contributed to the suspension culture. In addition, a chemically defined medium (selected by screening various prototype media) led to increased FMD virus production yields in the batch culture, even at a cell density of only 3.5 × 10⁶ cells/mL. The suspension BHK-21 cell culture could be expanded to a 200 L bioreactor from a 20 mL flask, which resulted in a comparable FMD virus titer. This platform technology improved virus productivity, indicating its potential for enhancing FMD vaccine production.

Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.