Development and optimization of the production process of a recombinant human protein by high-throughput techniques. Sviluppo e ottimizzazione del processo di produzione di una proteina umana ricombinante attraverso l'utilizzo di tecniche high-throughput.

Most biotechnology unit operations are complex with numerous process variables, feed material and raw material attributes that play significant impact on the performance of a process. Design of Experiment (DoE)-based approach together with HTPD (High-Throughput Process Development) techniques offer a solution to this issue allowing for an efficient estimation of effects and interactions by employing a minimal number of experiments. In the first part of the PhD work the HTPD approaches combined to statistical software were adopted in order to optimize a production process of a 340 kDa therapeutic recombinant protein expressed in PER.C6 cell line. The optimization of the purification process was performed by increasing the yield and by reducing the HCPs, residual DNA and viral contamination in order to obtain a fully compliant GMP process. This was achieved by introducing an arginine washing step during the capture on AEX resin and by reducing the concentration of phosphate in elution buffer in the intermediate step on hydroxyapatite, preventing the co-elution of the protein of interest and rDNA. A decrease of rDNA was then obtained by a membrane AEX step (by flow-through mode). Finally, two viral reduction steps (by thermal inactivation and filtration) were used in order to satisfy ICH Q5A guidelines. In the second part of the work, the DoE and HTPD technology were used as a “tool box” inside a step by step “decision tree” in order to lead the experimenter in a well defined path with the purpose of purifying and formulate any protein of interest. The protein used to test the procedure was a 13 kDa protein expressed in E. coli as inclusion bodies. The novel process allowed to obtain a pure and active preparation of the protein compliant to GMP requirements.