Development of Cleaning-in-Place Procedures for Protein A Chromatography Resins using Design of Experiments and High Throughput Screening Technologies

Abstract: Robust and efficient cleaning procedures for protein A chromatography resins used for production of monoclonal antibody based biopharmaceuticals are crucial for safe and cost efficient processes. In this master thesis the effect of different cleaning regimes with respect to ligand stability of two protein A derived media, MabSelectTM and MabSelect SuReTM, has been investigated. A 96-well format has been used for preliminary screening of different cleaning agents, contact times and temperatures. NaCl as a ligand stabilizer during cleaning-in-place (CIP) was also included as a parameter. For optimal throughput and efficiency of screening, Rectangular Experimental Design for Multi-Unit Platforms; RED-MUP, and TECAN robotic platform have been utilized. For verification of screening, selected conditions were run in column format using the parallel chromatography system ÄKTAxpressTM. In the efficiency study, where a manual preparation of CIP solutions was compared with an automated mode performed in TECAN, the total process time ended up at eight hours versus three days respectively. However, the time measured included the learning process for the TECAN platform and for further preparations the automated mode is the superior choice. The study confirmed the higher alkaline stability of MabSelect SuRe compared to MabSelect. After exposure to 0.55 M NaOH during 24h MabSelect SuRe still retained 90% of the initial capacity. In contrast MabSelect had 60% of the initial binding capacity. When CIP with 10 mM NaOH was performed at 40 °C MabSelect reduced its capacity by half while MabSelect SuRe still had a binding capacity of 80%. The 96-well screening showed that an addition of NaCl during CIP had a significant positive effect on the stability of MabSelect, but needs to be verified on column format. The correlation between results from screening in 96-well filter plate and column format was good.