Separation Of Anti-HER2 And Anti-IL-12/23 Monoclonal Antibodies By Hydrophobic Charge-induction Chromatography

Antibodies as the important therapeutic drugs have a broad market demand. The typical downstream process with Protein A (ProA) affinity chromatography has some limitations. It is necessary to develop the economical and efficient separation methods and new resins for antibody purification. Hydrophobic charge-induction chromatography (HCIC) as new bioseparation technology has some advantages in the adsorption and separation for antibodies. However, the processes and the function ligands of HCIC resins still need to be improved. With two monoclonal antibodies (mAbs) as the target, this work focused on HCIC processes to purify antibody from the CHO cell culture supernatant, including adsorption properties, optimization on the separation conditions, and the comparison of commercial and home-designed HCIC resins. New separation processes would be developed with high efficiency for antibody purification.Firstly, the typical commercial HCIC resin MEP-HyperCel was studied for the separation of anti-HER2 mAb from the CHO cell culture supernatant. The stability of anti-HER2 mAb in the culture broth was investigated and the adsorption onto MEP HyperCel at different pH was investigated. The adsorption capacities were high at the range of pH 6-9, but decreased significantly under acidic condition. The dynamic binding capacity of anti-HER2 mAb was determined with a packed bed, and the loading and elution conditions were optimized. After the optimization on the separation conditions, anti-HER2 mAb could be purified from cell culture supernatant with high efficiency. The purity reached 94.6%, and the yield was about 0.1mg/(ml feedstock).Secondly, new HCIC resin ABI with 5-amino-benzimidazole (ABI) as the ligand was used for the purfication of anti-IL-12/23 mAb from the CHO culture supernatant. The adsorption and separation performance were investigated and compared with MEP HyperCel. It was found that ABI resin had a good adsorption capacity for mAb, and the mass transfer in ABI resin was significantly faster than MEP HyperCel. The influence of flow rate on the dynamic binding capacity of ABI was slight, which meant that ABI is suitable for the separation with high velocity. Further the process was optimized, including loading and elution conditions, to separate anti-IL-12/23 mAb from the CHO culture supernatant. The purity and recovery were 98.6% and 93.0%, respectively, which was better than that with MEP HyperCel.Finally, home-designed HCIC resin with tryptophan-5-amino-benzimidazole (W-ABI) as the functional ligand was tested for the purification of anti-IL-12/23 mAb from the CHO culture supernatant. It was found that pH had a significant effect on the adsorption properties. W-ABI resin had high adsorption capacity for IgG at neutral pH and low adsorption at acidic pH. In addition, the dynamic binding capacity with W-ABI was little affected by the flow rate and high dynamic capacity could be maintained even at high flow rate. After the optimization of elution conditions, anti-IL-12/23 mAB could be purified from the cell culture supernatant with high purity of 97.2% and recovery of 93.3%. The removal of host cell protein (HCP) was better than that with MEP HyperCel, and the revcovey was higher than that with Protein A affinity resin, which showed a good potential for the applications.Aiming to the efficient separation of mAb from mammalian cell culture supernatant, HCIC processes were developed to improve the separation efficiency of mAb purification and the separation conditions were optimized. The performance of two home-designed HCIC resins were tested, and the results demonstrated the potential and feasibility of the new resins. HCIC processes would be a good alternative to Protein A affinity chromatography for mAb separationn.