Developing an upstream process for a monoclonal antibody including medium optimization

Background Monoclonal antibodies have been established as important therapeutics in cancer and autoimmune diseases. Hence, there is a growing interest in the production of monoclonal antibodies in pharmaceutical industry. In order to reduce timelines and costs of production the process and medium development is of central importance. Perfusion processes are well known to achieve higher productivities compared with batch or fed batch. Major advantages of perfusion culture are that you can keep optimal culture medium conditions for the cells and realize higher performance. However, obtaining high performance requires the combination of process optimization as well as a well-balanced concentrated culture medium. Selecting the best system also depends on the shear sensitivity of the cell line, the robustness of the process and the scale used. In upstream processing batch, fed batch and perfusion mode were applied. Design of Experiments (DoE) was used to develop a feed protocol for fed batch cultivations. In shake flask experiments the influence of temperature, osmolality, and pH to improve antibody yield was examined. In a further study we compared different cell retention systems with regard to achieve high viable cell densities in a short time like required for a seed train application. The best results were achieved with the ATF system with cell densities up to 1.3 × 10 cells/ml and 4 fold improved product concentration compared to batch culture. Materials and methods A CHO cell line producing the antibody G8.8 against Epithelial Cell Adhesion Molecule (Ep-CAM) was employed for the experiments performed in this study. The fermenters were Sartorius BBI Twin-System (2and 5 L culture volume). We compared five different retention systems: SpinFilter (Sartorius BBI Systems), Cell Settler (Biotechnology Solutions), Centritech Lab III (Pneumatic Scale), Biosep (Applikon) and ATF (Alternate Tangential Flow; Refine Technology). The cell count was performed with CEDEX cell counter (Roche Diagnostics). The monoclonal antibody was quantified with HPLC-method using Protein A-column. Design of Experiments (DoE) was used to develop a feed protocol for Perfusion cultivations. In shake flask experiments we examined the influence of temperature, osmolality, and pH to improve antibody yield.