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The 0.05). To further determine the efficiency of the Lomustine (CeeNU) model, large-scale experiments (30 and 250-L bioreactors) were conducted. protein. The effect of culture temperatures were investigated using the biphasic culture system. BoxCBehnken 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 37C to 30C during the late logarithmic phase and 30C to 28C 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. Introduction Nowadays, recombinant Fc-fusion protein production presents an attractive technique for development of new protein-based therapies against several chronic diseases including rheumatoid arthritis, platelet disorders and psoriasis [1, 2]. The Fc domain of such therapeutics, responsible for the hybrids prolonged half-life, is commonly coupled to a therapeutic protein and extends a protein’s half-life via the Fc receptor recycling mechanism [3, 4]. Chinese hamster ovary (CHO) cells are the preferred host expression system for Fc-fusion protein production whilst six out of nine Fc-fusion molecules on the market are expressed in CHO cells [1]. This is mainly due to the ability of CHO cells to perform complex post-translational modifications (PTMs). These modifications are crucial for full function of therapeutic products. The abolishment of PTMs may disrupt folding pathway [5]. Protein misfolding can be SMOH induced by a number of culture process parameters such as temperature, pH or protein Lomustine (CeeNU) biochemical modifications, including deamidation, oxidation and glycation [6]. It is further reported that misfolded proteins are produced during cell culture process and it can consist of more than 50% impurities at the end of the cell culture process, which leads to considerable yield decrements in commercial-scale manufacturing [7]. Furthermore, due to the biological activity and safety concerns, regulatory agencies mandate that recombinant therapeutic proteins should be free of impurities that can trigger immunogenicity. So, one of the major complicating factors in large-scale product development is elimination of product and process related impurities. Previous studies indicated that misfolded proteins might maximize the risk of immunogenic signal, explaining why reducing misfolded isomers of therapeutic proteins, as one of the protein-related impurities, is one of the main goals to build quality in final product [8]. Upstream process contains some operating parameters that if being optimized, they will contribute to high product yield Lomustine (CeeNU) while meeting the quality specifications. Amongst all the parameters, temperature has a significant impact on accurate protein folding. In addition to potential benefits on protein folding, applying sub-physiological temperatures during cell culture process especially in production phase has also been reported to increase the cell-specific productivity [9]. Marchant and his colleagues reported the cell-specific productivity (qMab) of IgG4 monoclonal antibody (cB72.3) as increased by 50% after temperature shift to 32C from 37C during the stationary phase of growth [10]. It is quite in contrast with the improved production of anti-4-1BB antibody, which is highly suppressed at lower temperature (30, 33 degrees C) [11]. Furthermore, the time point of culture temperature shifting Lomustine (CeeNU) can also affect cell proliferation rate and productivity. These two parameters should be set in a way to effectively decrease the protein misfolding while minimizing the effect of temperature downshift on cell growth and viability. The culture temperature can be switched gradually or in discrete intervals while the culture shifts from growth phase to production phase [12]. The gradually decrease in temperature may result in a much better performance of cells regarding growth rate and viability while improving the protein folding at lower culture temperatures. To investigate the role of culture temperature on product yield and production of misfolded protein in a specific CHO cell line, we first examined the effect of low culture temperatures in the range of 28C32C. Lowering the temperature had been shown to be beneficial for protein folding, while being detrimental for production yield. To provide insight into the conditions with higher yield and quality, two-step temperature downshift was then employed. The conventional method of process optimization, one factor-at-a-time, is time-consuming, expensive, and often leads to misinterpretation of results when interactions between different components are present. Statistical experimental designs minimize the error.