Affinity Precipitation of Monoclonal Antibodies Using Stimuli Responsive Smart Biopolymers: Methods Development and Process Considerations

Development of novel protein purification approaches is crucial for sustaining the continued growth of the biopharmaceutical industry. Elastin-like polypeptides (ELPs) are stimuli responsive smart biopolymers which undergo reversible phase transition above a certain critical temperature called the inverse transition temperature. This property has made ELPs attractive tools for protein purification. This work provides a detailed investigation into the development of a robust and scalable monoclonal antibody (mAb) affinity precipitation process using elastin-like polypeptides fused to the mAb binding Z domain (ELP-Z).;A multidimensional high-throughput screening (HTS) protocol is developed and employed to investigate the effects of a variety of operating conditions on mAb yield and aggregation during the affinity precipitation of pure model mAbs. HTS is initially employed to determine conditions for the capture and co-precipitation of mAbs at high yields by the ELP-Z. A subsequent HTS screen is then employed to investigate mAb elution from the ELP-Z-mAb complex. The results suggest that high mAb yields with low aggregate content can be achieved under mild elution conditions (pH 4.2) at room temperature. The findings from the HTS studies are further employed to guide mAb purification from an industrial mAb harvest feed. The process is shown to result in more than 2 logs of host cell protein and more than 4 logs of DNA impurity clearance from the harvest feed, comparable or superior to Protein A chromatography performance for that mAb. Performance is maintained for mAb final elution concentrations of up to 20 g/l and effective ELP-Z regeneration and its reusability are also demonstrated.;Finally, a proof of concept scalable mAb affinity precipitation process is developed using scaled down filtration techniques. Tangential flow filtration (TFF) is initially employed for the recovery of the precipitates formed during the process. While the precipitates formed by the ELP-Z-mAb complex can be recovered using TFF at high throughputs, the ELP-Z precipitates foul the membrane significantly. Confocal microscopy is used to gain further insights into the nature of precipitation. A normal flow filter train is then successfully employed for recovering the ELP-Z precipitates at high throughputs. The results presented in this thesis clearly demonstrate the potential of the ELP-Z based affinity precipitation process as a next generation unit operation for industrial mAb bioprocessing.