Expanded Bed Adsorption With Hydrophobic Charge-Induction Ligand And The Applications For Antibody Separation

Antibodies are the most important bioproducts which are in great demand in the medical and research fields. Typical antibody separation processes usually involve Protein A affinity chromatography. However, this technology shows some limitations, such as high cost, low capacity, and difficult elution and regeneration. Therefore, it is necessary to develop new methods of antibody separation for industrial applications. Expanded bed adsorption (EBA) and hydrophobic charge-induced chromatography (HCIC) are two innovative bioseparation technologies that can be used in antibody separation. EBA can capture target proteins directly from the crude feedstock without any pretreatments such as solid-liquid separation, which simplifies the process and improves the separation efficiency. HCIC combines multiple molecular interactions to enhance the protein binding with high adsorption capacity and strong salt tolerance. In this thesis, HCIC and EBA were combined to develop a new separation technology: hydrophobic charge-induction EBA and its application was discussed using human immunoglobulin G (hIgG) and bovine immunoglobulin G (bIgG) as model target proteins.Two HCIC resins for EBA were prepared with Streamline agarose beads containing quartz densifer as matrix and 4-mercapto-ethyl-pyridine (MEP) (S-MEP) and 5-aminobenzimidazole (ABI) (S-ABI) as functional ligands, respectively. The adsorption performance of two resins was investigated and S-MEP and S-ABI both showed pH-dependent and salt-tolerant adsorption properties for bIgG and hIgG. S-ABI had higher dynamic adsorption capacity than S-MEP. In addition, the expanded bed filled with S-ABI was stabile with low degree of axial mixing. The optimized expansion factor for EBA was 1.8 (operating velocity 195 cm/h) and the dynamic adsorption capacities for bIgG and hIgG were 3.71 and 28.58 mg/ml settled resin, respectively.In order to improve flowing properties and dynamic adsorption capacity of S-ABI, tungsten carbide/agarose composite beads were used as the matrix for MEP and ABI HCIC resins (named T-MEP and T-ABI, respectively). Similar to S-MEP and S-ABI, T-MEP and T-ABI also showed pH-dependent and salt-tolerant properties for bIgG and hIgG adsorption. However, higher operating velocity and higher dynamic adsorption capacity were obtained with T-MEP and T-ABI. The expanded bed with T-ABI was stable with low axial mixing. The best expansion factors for EBA were 2.0 and 2.2 (corresponding to 889 and 1050 cm/h). The dynamic adsorption capacities for bIgG and hIgG reached 5.49 and 21.38 mg/ml settled resin, respectively.T-ABI was further tested for antibody separation with IgG/BSA mixture as the model system. Effects of loading pH, elution pH, expansion factor and loading volume were evaluated. The optimized conditions for bIgG separation were:loading at pH 8.0, elution at pH 3,5,2 times of settled bed volume for loading. The separation conditions for hIgG were:loading at pH 7.0, elution at pH 4.0 or 4.5,3 times of settled bed volume for loading. The expanded bed with T-ABI was stable for IgG separation at a wide range of operation velocities with the best expansion factor of 2.0. In addition, there was competition between IgG and BSA. When the loading volume increased, BSA adsorbed on resins was partly replaced by IgG and resulted in the IgG purity increase. Bovine whey and CHO culture broth were further separated with good separation performance. The purity and recovery of bIgG from bovine whey were 90.6% and 78.2% with purification factor of 19.3. The purity and recovery of MAB from CHO culture broth were 97.7% and 72.6% with purification factor of 7.5 at elution pH 4.5, which were 93.7% and 79.4% with purification factor of 7.2 at elution pH 4.0.In-bed sampling and mathematical models were adopted to characterize axial distribution of resin beads and protein adsorption. The results indicated that the model agree well with the experimental data. The particle size decreased but the bed voidage increased with the increase of bed height. The bottom zone of expanded bed played an important role on the protein adsorption and the adsorption of other zones decreased with the increase of bed height. It was verified that the model could predict the axial distribution of protein adsorption. In addition, effects of different factors including adsorption parameters (Qm, Kd, Dp and Co), operating condition parameters (Dcolumn, H0,ρL, μL and EF) and matrix parameters (Dmean, σ and ρp) on bed expansion, particle size axial distribution, bed voidage and protein adsorption were further analyzed. The results could be used to guide new resins and separation process design for EBA.In this thesis, combining HCIC and EBA, new type of expanded bed resins were developed with quartz/agarose beads and tungsten carbide/agarose composite beads as matrix and MEP and ABI as functional ligands. The adsorption performances for IgG were evaluated, and the separation of IgG from real feedstock was realized. It was demonstrated that the new HCIC-EBA technology is feasible for antibody separation and would be further studied for more applications.