Implementation of Design of Experiments (DOE) for Optimization of Feeding Strategy and Glyco-Engineering of Trastuzumab Biosimilar

Fed-batch cell culture is the most commonly used process for antibody production in biopharmaceuticalindustries. Basal media, feed, feeding strategy and glycanstructures are always among the most important concerns during process development and optimization. In this study, first, a traditional screening study was performed to identify the top media/feed combinations by evaluating the cell culture performance including cell growth and protein titre. Optimization of the process was also performed using response surface methodology in order to find the most optimum feeding strategy and glucose set point regarding final titre of the recombinant monoclonal antibody being produced in Chinese hamster ovary cellline. The focus of this study is not only on titre, but also on product quality and comparability especially protein glycosylation. The prediction model of product titre as a function of feeding percentage and glucose set point was successfully applied for the second set of experiments that was performed for glycanimprovement. Statistical design of experiments was applied to determine the most important factors and their effects on galactosylated and afucosylated glycans. Uridine, manganese, galactose and fucosyltransferaseinhibitor were chosen to evaluate if their presence can affect glycansand to obtain their best combination for fed-batch culturesupplementation. We determined that 2.5 % daily feeding combined with maintaining the glucose set point on 2.5±0.2 g/L could achieve final titre of 2.5±0.1 g/L. Galactosylation of antibody was increased about 25% using MnCl2 and galactose while afucosylation was increased about 8% in presence of fucosyltransferaseinhibitor. Galactose and Mn2+ led to a shift from G0F to G1F and presence of Fucosyltransferaseinhibitor caused to an increase in G0 compared to its absence. These results demonstrated that supplementation of culture with all these components can provide exact control of antibody galactosylation and fucosylationwith minimal impact on culture characteristics and product quality attributes. Subsequently, validation experiments were also carried out in 5L STR bioreactors which showed that similar results could be achieved in bioreactors compared to shake flasks regarding both titre and quality.