Sustainable Production of Recombinant FGF2 in Cell Culture Spent Media using Engineered Lactococcus lactis: Modelling and Process Integration

Spent mammalian cell culture media are commonly discarded despite having residual nutrients that could be repurposed for microbial biomanufacturing. Fibroblast growth factor 2 (FGF2) is a major cost contributor in serum-free cell culture media, motivating the development of economical production strategies. Here, we investigated the use of fortified spent cell culture media for FGF2 production using Lactococcus lactis, a GRAS microorganism, and integrated statistical optimization, kinetic modeling, and continuous bioprocessing. A Box-Behnken design identified 10g/L glucose, 35 °C cultivation temperature, and 100ng/mL nisin as optimal conditions for enhancing intracellular and secreted FGF2 production. Subsequently, kinetic model comprising of ordinary differential equations was developed to describe temporal profiles of intracellular & secreted FGF2, glucose, lactate, and biomass. The model integrated Monod-type kinetics for growth and substrate consumption, and Luedeking-Piret kinetics to account for FGF2 production. Leveraging this model, we demonstrated that model could guide selection of dilution rate to maximize FGF2 production. To advance toward continuous manufacturing, chemostat cultivation was coupled with depth and crossflow filtration to enable real-time cell separation and FGF2 concentration. The concentrated FGF2 demonstrated comparable bioactivity to commercial FGF2 in proliferating Anguilla japonica (Japanese eel) pre-adipocytic cells. Overall, our study demonstrated utilization of design of experiments (DoE) alongside kinetic modelling to optimize bioprocess conditions, achieving FGF2 production of 16mg/L and 396µg/L titres for intracellular and secreted forms, respectively. The circular bioprocess, use of predictive modelling and integration of continuous upstream-downstream operation provides an opportunity for growth factor manufacturing from L. lactis.