| Mixed-mode chromatography (MMC) is a novel bioseparation technique. The mixed-mode ligands have multiple-binding modes, such as hydrophobic interaction, electrostatic interaction, and hydrogen bond. MMC has been used in the field of antibody purification with the advantages of high adsorption capacity, good selectivity and mild elution conditions. Based on our previous works, new MMC resins KB-ABI with 5-Aminobenzimidazole (ABI) as the functional ligand compared and polymethyl acrylate beads as the matrix. Compared with the commercial MMC resins Nuvia cPrime with p-aminohippuric acid (PAH) as the functional ligand, the adsorption properties of immunoglobulin G (IgG) and bovine serum albumin (BSA) were investigated under different conditions. The adsorption and transport processes were visualized by confocal laser scanning microscopy (CLSM). In addition, the separation of IgG from IgG/BSA mixture and the purification of monoclonal antibody (mAb) from CHO cell culture supernatant were evaluated.Main results were listed as follows:(1) With bovine IgG (bIgG) and BSA as the model proteins, the adsorption properties on Nuvia cPrime resins were investigated, including static adsorption, adsorption and elution behaviors in the column. It was found that pH and salt addition could significantly affect the adsorption behaviors for both proteins. The maximum difference of adsorption capacity for two proteins appeared at pH 6.0. The adsorption capacities of both proteins decreased with the increase of NaCl concentration while "U-shaped" trends were found with the increase of (NH4)2SO4 concentration. The results indicated that the adjustment of pH and salt addition could improve the separation of bIgG and BSA. The suitable separation conditions would be loading at pH 6.0 with 0.8 M (NH4)2SO4 and elution at pH 8.0 with 1.0 M NaCl.(2) With human IgG (hIgG) and BSA as the model proteins, the co-adsorption behaviors of two proteins on Nuvia cPrime resins with various mass ratios were studied. The result indicated that there were some competitive relations between two proteins and hIgG dominated the adsorption in the binary-component system. The molar ratio of hIgG to BSA in the adsorbent was 10 times higher than that in the liquid at pH 6.0. Based on the parameters correlated from the adsorption experiments with single component, the binary-component adsorption behaviors were predicted by the Extended Langmuir (EL) and Extended Langmuir-Freundlich (ELF) adsorption models. The calculation values fitted well to the experimental data.(3) New MMC resin KB-ABI was prepared with polymethyl acrylate beads as the matrix and 5-Aminobenzimidazole (ABI) as the functional ligand. The ligand density could reach 195 μmol/mL after the optimization of the activation and ligand coupling conditions. With hIgG and BSA as the model proteins, the static adsorption properties on KB-ABI resins under different conditions were investigated. Typical pH-dependent and salt-tolerant adsorption properties were found for both proteins. The maximum adsorption capacity of hIgG was found at pH 8.0, while the adsorption capacity of BSA decreased as pH increased. In addition, the co-adsorption behaviors of hIgG and BSA at different mass ratios were investigated, and the competitive adsorption behavior were found, especially at pH 5.0 and 8.0. The co-adsorption behaviors were also predicted by EL and ELF models, and the predictions agreed well with experimental data. The adsorption behaviors in the column revealed that loading at pH 8.0 with 0.2 M NaCl and elution at pH 5.0 could effectively separate hIgG from the mixture of hIgG and BSA.(4) The adsorption kinetic behaviors were investigated with two resins and two proteins at different pH, including the single and binary-component system. The results indicated the competitive adsorption behaviors between two proteins. For the binary adsorption system, the BSA adsorption capacity has a maximum value at 40 min and then decreased gradually to the equilibrium adsorption capacity. This phenomenon demonstrated that there was a competitive and replacement process between hIgG and BSA. Moreover, the single component kinetic adsorption was fitted by line drive force model (LDF) and the binary-component dynamic adsorption was well fitted by the modified LDF model. The surface diffusion coefficient (Ds) was obtained. It was found that Ds values of hIgG changed slightly with pH, and different trends were found for BSA on two resins tested.(5) The protein mixture of IgG and BSA at mass ratio of 1:4 and CHO cell culture supernatant were used as the feedstock, the separation efficiencies of Nuvia cPrime and KB-ABI resins were evaluated. The purity, recovery of antibody and the removing of host cell protein (HCP) were compared. For Nuvia cPrime, the purity and recovery of IgG separated from IgG/BSA mixture reached 99.8% and 92.7%, while the purity and recovery of mAb separated from cell culture reached 92.8% and 82.5%. For KB-ABI, the purity and recovery of IgG reached 99.7% and 94.6%, and the purity and recovery of mAb reached 94.9% and 92.5%, which was comparable with Protein A affinity resin. For the HCP clearance, the performance of KB-ABI was comparable with Protein A affinity chromatography and much better than that of Nuvia cPrime, which indicated a good application potential.(6) In order to reveal the adsorption mechanism, CLSM was used to investigate the adsorption and transport process of two proteins on two MMC resins, Nuvia cPrime and KB-ABI. It was verified that there was slightly impact on the protein adsorption behavior after labeling with dyes. The single component adsorption on two resins at different pH was studied firstly by CLSM. The results indicated that the adsorption mechanism was the combination of porous diffusion and surface diffusion. In addition, the fluorescence intensities were correlated quantitatively to the adsorption capacities of proteins. Secondly, the binary component adsorption behaviors were also investigated by CLSM. The overshoot profile formation mechanism and competitive adsorption behavior were discussed with the results of CLSM. Finally, the desorption and sequence adsorption behaviors were investigated. The results revealed that the surface zone of adsorbent had stronger binding force, and there were some differences between Nuvia cPrime and KB-ABI in the protein adsorption processes.In this thesis, the adsorption properties of two new MMC resins were focused, and the influences of pH and salt addition were studies. Based on the results of adsorption behaviors, the separation and purification of antibodies were optimized. Furthermore, CLSM was introducted to study the adsorption mechanisms of MMC resins. The results obtained in this thesis would be useful for the applications of antibody purification with MMC processes. |
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