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.

细胞培养工艺优化是应对制药制造面临的大多数挑战的关键解决方案。大规模生产治疗性蛋白质时遇到的主要问题之一是错误折叠的蛋白质生产。错误折叠的治疗蛋白的积累是免疫原性信号，也是最终产品免疫原性的危险因素。本研究的目的是对三相温度变化和时间进行统计优化，以提高正确折叠的 Fc 融合蛋白的产量。使用双相培养系统研究培养温度的影响。然后使用 Box-Behnken 设计来计算最佳转换的温度和时间。响应面方法显示，在对数后期从 37°C 到 30°C 以及在稳定中期从 30°C 到 28°C 的两步温度转变中，实现了低水平错误折叠蛋白质的最大产量。优化条件给出了最佳结果，蛋白滴度为1860 mg L ^{-1 ，错误折叠水平为24.5%。}验证实验在最佳条件下进行了 3 个重复，蛋白质错误折叠水平降低了两倍，而生产率提高了约 1.3 倍。在最佳条件下250 L生物反应器的规模化生产也验证了模型的有效性和准确性。结果表明，利用两步温度变换，生产率和目标蛋白的质量同时得到提高。该模型可以成功应用于其他产品。