Background: Drastic pH drop is a common consequence of scaling up a mammalian cell culture process, where it may affect the final performance of cell culture. Although CO2 sparging and base addition are used as common approaches for pH control, these strategies are not necessarily successful in large scale bioreactors due to their effect on osmolality and cell viability. Accordingly, a series of experiments were conducted using an IgG1 producing Chinese Hamster Ovary (CHO-S) cell culture in 30 L bioreactor to assess the efficiency of an alternative strategy in controlling culture pH.   Methods: Factors inducing partial pressure of CO2 and lactate accumulation (as the main factors altering culture pH) were assessed by Plackett-Burman design to identify the significant ones. As culture pH directly influences process productivity, protein titer was measured as the response variable. Subsequently, Central Composite Design (CCD) was employed to obtain a model for product titer prediction as a function of individual and interaction effects of significant variables.   Results: The results indicated that the major factor affecting pH is non-efficient CO2 removal. CO2 accumulation was found to be affected by an interaction between agitation speed and overlay air flow rate. Accordingly, after increasing the agitation speed and headspace aeration, the culture pH was successfully maintained in the range of 6.95-7.1, resulting in 51% increase in final product titer. Similar results were obtained during 250 L scale bioreactor culture, indicating the scalability of the approach.   Conclusion: The obtained results showed that pH fluctuations could be effectively controlled by optimizing CO2 stripping.

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.

2Fed-batch cell culture is the most commonly used process for antibody production in 3 biopharmaceutical industries. Basal media, feed, feeding strategy and glycan structures are always 4 among the most important concerns during process development and optimization. In this study, 5 first, a traditional screening study was performed to identify the top media/feed combinations by 6 evaluating the cell culture performance including cell growth and protein titre. Optimization of the 7 process was also performed using response surface methodology in order to find the most optimum 8 feeding strategy and glucose set point regarding final titre of the recombinant monoclonal antibody 9 being produced in Chinese hamster ovary cell line. The focus of this study is not only on titre, but 10 also on product quality and comparability especially protein glycosylation. The prediction model 11 of product titre as a function of feeding percentage and glucose set point was successfully applied 12 for the second set of experiments that was performed for glycan improvement. Statistical design 13 of experiments was applied to determine the most important factors and their effects on 14 galactosylated and afucosylated glycans. Uridine, manganese, galactose and fucosyltransferase 15 inhibitor were chosen to evaluate if their presence can affect glycans and to obtain their best 16 combination for fed-batch culture supplementation. We determined that 2.5 % daily feeding 17 combined with maintaining the glucose set point on 2.5±0.2 g/L could achieve final titre of 2.5± 18 0.1 g/L. Galactosylation of antibody was increased about 25% using MnCl 2 and galactose while 19 afucosylation was increased about 8% in presence of fucosyltransferase inhibitor. Galactose and 20 Mn 2+ led to a shift from G0F to G1F and presence of Fucosyltransferase inhibitor caused to an 21 increase in G0 compared to its absence. These results demonstrated that supplementation of culture 22 with all these components can provide exact control of antibody galactosylation and fucosylation 23 with minimal impact on culture characteristics and product quality attributes. Subsequently, 24 validation experiments were also carried out in 5L STR bioreactors which showed that similar 25 results could be achieved in bioreactors compared to shake flasks regarding both titre and quality. 26 Keywords: Trastuzumab biosimilar, feeding strategy optimization, design of 27 experiments (DOE), galactosylation, afucosylation 3 28 Abbreviations 29 mAb: Monoclonal antibody; DOE: design of experiment; RSM: response surface 30 methodology; CDC: complement dependent cytotoxicity; ADCC: antibody dependent cell 31 cytotoxicity; CQA: critical quality attributes; Fc: fragment crystallizable; FTI: fucosyl transferase 32 inhibitor; CHO: Chinese hamster ovary; HER2; human epidermal growth factor receptor 2; CCD: 33 central composite design; ANOVA: analysis of variance; HPLC: high pressure liquid 34 chromatography; STR : stirred tank reactor; DO: Dissolved oxygen; VCD: viable cell density; 35 VCC: viabl...