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

Abstract

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.

Fig 1. Kinetics of cell growth.

Cell density of Fc-fusion producing CHO cell line (continues lines). Viabilities of Fc-fusion producing CHO cell line (dashed lines). Control condition was carried out at 37°C. (□, Control culture; Δ, shift to 32°C; ○, shift to 31°C; ♦, shift to 30°C; *, shift to 29°C; ▲, shift to 28°C. Vertical dashed line indicates the time of temperature shift.

Fig 2

a) Lactate levels (mM) and b) daily glucose uptake rate (g/L/day) for fed-batch bioreactor cultures.

Fig 3

Response surface for Fc-fusion protein titer and protein misfolding level as a function of temperature (°C) and first temperature shift time (h) (a and d), temperature (°C) and second temperature shift time (h) (b and e) and first temperature shift time (h) and second temperature shift time (h) (c and f). A: temperature (°C), B: first temperature shift time (h) and C: second temperature shift time (h).

Fig 4. Elution profile of Fc-fusion protein on a butyl NPR HIC-HPLC column.

Optimized condition (three-phasic cultivation) results in a decrease of post-main-peak (P2) compared to the control condition (P1). Control condition was carried out at physiological temperature (37°C).