OPTIMIZATION OF CLONE SELECTION PLATFORM FOR PRODUCTION OF RECOMBINANT PROTEINS

Sammanfattning: Background  Biologics are type of proteins that can be produced by expression in a foreign host for medical healthcare applications in humans. Monoclonal antibodies (mAbs) are type of biologics that are designed to specifically bind to one target, with a purpose to inhibit a certain molecule that could be associated with a disease process. Usually, the usage of mammalian cells as expression hosts are generally preferred for biologics production. Among the available choices of mammalian host cells, Chinese hamster ovary (CHO) cell line is considered to be the initial choice for biologics production due to their compatibility with human proteins and their scalability to grow in large scale.  Aims This project aims to establish a clone selection platform, to identify the best producing clone from a specific cell line and its clones from metabolic point of view (CHO-SDI-X, CHO-SDI-Y, CHO-SDI-Z). These cell lines were established through site-directed integration.  Specifically, the goal of the project is to:  ·   evaluate cell culture kinetics and metabolic profiles for each cell line in batch and fed-batch culture. ·   identify the most adequate feed rate for future clone selection platform based on these cell lines and fed-batch regime. Methods/Materials  Cells were thawed and passaged (seed trains) initially (target initial and end viable cell densities for each seed-train step were 0.5(±0.1) and 4 (±1) MC/mL, respectively), before cultured into batch and fed-batch mode. Secondly, cells were grown in batch mode and each cell line were grown in three biological replicates. Finally, during fed-batch mode, cell specific feed rate strategy was implemented as the feed strategy for the experiment. In addition, 3 different feed rates (0.5, 0.75 and 1.125 pL/cell/day) were tested to identify the adequate one for the cells in addition to measuring the produced metabolites. Samples were taken daily from each culture flask in both batch and fed-batch to measure viable cell density, viability, and metabolites (Gluc, Lac, Gln, Glu, NH4+).  Results The data generated from the batch and fed-batch culture revealed differences in the cell kinetics and metabolic profile between CHO-SDI-Z and the other two cell lines. Also, during batch experiment, different growth phases of each cell line including the exponential growth phase was identified. In addition, after screening different feed rates and testing the cells in fed-batch mode it was identified that 0.75 pL/cell/day was the most adequate feed rate, and 1.125 pL/cell/day drove the cells to die/crash suddenly after 12 days of continuous growing.  Conclusions: As a conclusion, the starting point of fed-batch regime were determined for each tested cell line. Also, the driving factors for the end of the exponential phase and end of stationary phase have been identified. Furthermore, it was concluded that CHO-SDI-X and CHO-SDI-Y had similar cell culture kinetics and metabolic profiles, while CHO-SDI-Z had different demeanour. Finally, the optimal feed rate was identified.