Bioprocess Intensification Using Perfusion Culture for Recombinant Protein Production from Lactococcus lactis

Secretion of recombinant proteins from microbial cell factories is a promising strategy for recombinant protein biomanufacturing as it simplifies downstream processing. In this study, we sought to intensify the Lactococcus lactis high-cell-density culture, using basic fibroblast growth factor 2 (FGF2) as a model recombinant protein. We implemented a perfusion strategy using tangential flow filtration for cell retention, allowing continuous removal of inhibitory metabolites while replenishing fresh nutrients. When conventional 2×GM17 medium was used, the approach outperformed batch cultivation, achieving a 5.2-fold increase in biomass and a 2.6-fold increase in secreted FGF2, reaching a titer of 9000 µg·L⁻¹. Concurrently, we developed a bioprocess model for L. lactis grown in a fortified spent cell culture medium, enabling systematic exploration of operating conditions. A Pareto front was generated for FGF2 titer against media usage, and perfusion profiles balancing both competing objectives were identified. An experimentally selected operating point validated the model predictions, yielding a final OD₆₀₀ of 49.2 and FGF2 titer of 2166 µg·L⁻¹ FGF2, which were 8-fold and 5-fold higher than batch process respectively. Overall, this work demonstrates a perfusion-based intensification strategy for L. lactis and highlights the utility of model-guided process decision making to enhance productivity while reducing waste.