Therapeutic Fc fusion protein misfolding: A three-phasic cultivation experimental design.

Cell culture process optimization is a critical solution to most of the challenges faced by the pharmaceutical manufacturing. One of the major problems encountered in large-scale production of therapeutic proteins is misfolded protein production. The accumulation of misfolded therapeutic proteins is an immunogenic signal and a risk factor for immunogenicity of the final product. The aim of this study was the statistical optimization of three-phasic temperature shift and timing for enhanced production of correctly folded Fc-fusion protein. The effect of culture temperatures were investigated using the biphasic culture system. Box-Behnken design was then used to compute temperature and time of shifting optimum. Response surface methodology revealed that maximum production with low level of misfolded protein was achieved at two-step temperature shift from 37°C to 30°C during the late logarithmic phase and 30°C to 28°C in the mid-stationary phase. The optimized condition gave the best results of 1860 mg L-1 protein titer with 24.5% misfolding level. The validation experiments were carried out under optimal conditions with three replicates and the protein misfolding level was decreased by two times while productivity increased by ~ 1.3-fold. Large-scale production in 250 L bioreactor under the optimum conditions was also verified the effectiveness and the accuracy of the model. The results showed that by utilizing two-step temperature shift, productivity and the quality of target protein have been improved simultaneously. This model could be successfully applied to other products.