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.
Tao Liu,Dong-Qiang Lin,Qi-Ci Wu,Qi-Lei Zhang,Cun-Xiang Wang,Shan-Jing Yao
Hydrophobic charge-induction chromatography (HCIC) is a developing technology for antibody purification. To enhance the protein adsorption capacity, a novel polymer-grafted HCIC resin was developed, in which the surface-initiated activator generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) was explored as a controlled polymerization technique to reconstruct matrix structure and ligand distribution. Using poly(glycidyl methacrylate, GMA) as grafting polymer and 2-mercapto-1-methyl-imidazole (MMI) as functional ligand, poly(GMA)-grafted HCIC resins were prepared with series of grafting and ligand densities. Adsorption behaviors of human immunoglobulin G (hIgG) on the prepared resins demonstrated the necessity of controlling grafting and ligand density. Saturated adsorption capacity ( Q m ) and effective pore diffusivity ( D e ) reached the maximum under medium ligand density when the grafting density was kept constant. The highest Q m and D e values were found under the highest grafting density, which were 73% and 7.17 times higher than the non-grafted resin, respectively. Column breakthrough tests indicated that the dynamic binding capacity of the resin with optimized grafting density and ligand density was up to 34.6 mg/g when linear velocity was 300 cm/h, which was 86.3% higher than dextran-grafted resin. The resin was then used to separate hIgG from a protein mixture (hIgG/human serum albumin = 1:4), high purity (>99%) and recovery (>90%) of hIgG were found with 50-cycle reuses, which verified the selectivity and robustness of G-MMI resin prepared. In general, the surface-initiated AGET ATRP provides a controlled grafting strategy to improve protein binding capacity for chromatographic separation, and new resins developed have great potential in large-scale protein purification applications.
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.
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