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 the continuous removal of inhibitory metabolites while replenishing fresh nutrients. When conventional 2 × GM17 medium was used, the approach outperformed batch cultivation, achieving a 4.7-fold increase in biomass and a 3-fold increase in secreted FGF2, reaching a titer of 10,416 ± 892 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 ± 0.3 and FGF2 titer of 2166 ± 161 µg·L^-1 FGF2, which were 7.9-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.