The huge demand for the most promising biopharmaceuticals, monoclonal antibodies (mAb), has resulted in a need for more efficient and less costly downstream purification processes for mAb. The currently used Protein A Agarose is unattractive due to toxic ligand leakage and high cost. In this study, three novel small peptide ligands, DWHW, CEWW and HEYW, were designed based on the interactions with human immunoglobulin G (IgG) using molecular simulations. The effects of pH, ionic strength and flow rate on the binding capacities to IgG were investigated. The static and dynamic binding capacities were determined. The dissociation constant (Kd) of the DWHW resin was 1.1 × 10−5 M. The binding capacity of the DWHW resin was 24.5 mg ml−1, which is comparable to Protein A Agarose. The DWHW resin was able to purify IgG from cMEM and CHO cell culture supernatants with a purity of more than 95% under elution conditions of pH 9 (50 mM sodium borate buffer) and pH 3 (50 mM glycine–HCl buffer). The results indicate that the small peptide ligands, especially DWHW, can offer a potential alternative for mAb purification.
Hong-Fei Tong,Dong-Qiang Lin,Dong Gao,Xiao-Ming Yuan,Shan-Jing Yao
Hydrophobic charge-induction chromatography (HCIC) is a novel downstream bioprocessing technology for antibody purification and it has several advantages over traditional purification processes. However, its separation selectivity still needs to be improved. In this work, sodium caprylate (NaCA) was used as the selective modifier to improve IgG purification from serum albumin containing feedstock with a typical HCIC resin, MEP HyperCel. The effects of NaCA on the adsorption equilibrium of bovine serum immunoglobulin G (IgG) and bovine serum albumin (BSA), as well as the dynamic binding and displacement behaviors were investigated. The binding and elution behaviors of these two proteins in the column were studied. It was found that adding 50–75 mM NaCA in the liquid phase could effectively reduce the adsorption of BSA on the MEP resin, but the same treatment has little influence on the adsorption of IgG. Moreover, the mechanism of the competitive binding between caprylate and MEP ligands on the surface of BSA is discussed. It was found that by controlling NaCA addition in the loading or washing buffer, the process efficiency of IgG purification from BSA containing feedstock can be improved, and the purity of IgG could reach to over 98%. The results indicated that caprylate could be a promising albumin-selective modifier to improve the separation efficiency of antibodies with the HCIC process.
Hong-Fei Tong,Dong-Qiang Lin,Wen-Ning Chu,Qi-Lei Zhang,Dong Gao,Rong-Zhu Wang,Shan-Jing Yao
Hydrophobic charge-induction chromatography (HCIC) has advantages of high capacity, salt-tolerance and convenient pH-controlled elution. However, the binding specificity might be improved with multimodal molecular interactions. New ligand W-ABI that combining tryptophan and 5-amino-benzimidazole was designed with the concept of mutimodal charge-induction chromatography (MCIC). The indole and benzimidazole groups of the ligand could provide orientated mutimodal binding to target IgG under neutral pH, while the imidazole groups could induce the electrostatic repulsion forces for efficient elution under acidic pH. W-ABI ligand was coupled successfully onto agarose gel, and IgG adsorption behaviors were investigated. High affinity to IgG was found with the saturated adsorption capacity of 70.4 mg/ml at pH 7, and the flow rate of mobile phase showed little impact on the dynamic binding capacity. In addition, efficient elution could be achieved at mild acidic pH with high recovery. Two separation cases (IgG separation from albumin containing feedstock and monoclonal antibody purification from cell culture supernatant) were verified with high purity and recovery. In general, MCIC with the specially-designed ligand is an expanding of HCIC with improved adsorption selectivity, which would be a potential alternative to Protein A-based capture for the cost-effective purification of antibodies.
危险品化学品经营许可证（带存储）