Influences Of Pore Size And Ligand Density Of Chromatographic Resins On The Protein Adsorption And Antibody Separation

Chromatography is a widely used technique in protein separation, and its separation efficiency is largely affected by chromatographic resins. Research usually focuses on structures of functional ligands coupled onto resins, while the pore size of resin matrices and ligand density are less discussed. This thesis investigated ion-exchange chromatography (IEC) resin and hydrophobic charge induction chromatography (HCIC) re’ sins on their application in antibody separation. Series of diethylaminoethyl (DEAE) IEC resins and2-mercapto-l-methylimidazole (MMI) HCIC resins with different ligand densities and pore sizes were prepared. The adsorption performance of these resins was characterized with the influences of ligand density and pore size. The findings of this thesis can be summarized as:(1) Three cross-linked agarose gels with different pore sizes (Bestarose3.5HF,4FF and6FF) were used as the matrices. DEAE IEC resins and MMI HCIC resins with different ligand densities and pore sizes were prepared by the control of ligand coupling reactions. The results showed that the highest ligand density of DEAE resins could reach to110,280and410μmol/g gel, respectively, and that of MMI were50,110and130μmol/g gel, respectively.(2) The static adsorption behaviors of the DEAE and MMI resins were investigated using bovine immunoglobulin (bIgG) and bovine serum albumin (BSA) as model proteins. The adsorption isotherms obtained were fitted by Langmuir equation. The results showed that the adsorption capacity of DEAE increased with the increase of ligand density when the ligand density was relatively low, and it reached saturation then deceased slightly when the ligand density further increased. Meanwhile, the adsorption capacity increased with the decrease of pore size, as the specific surface area of the resins was higher with smaller pore size. A parameter N defined as the ratio of ligand density to the square of pore size was introduced to describe the integrative effects of pore size and ligand density, and it was found that the saturated adsorption capacity increased linearly with log (N). For MMI resins, the results showed that BSA and bIgG could not be adsorbed with low ligand densities, and resins with ligand density over15x10-12μmol/μm2were need for effective adsorption of BSA and bIgG(3) The dynamic adsorption performance for bIgG and BSA were investigated with the DEAE and MMI resins prepared. The adsorption kinetics curves were fitted with the pore diffusion model (PDM). For DEAE resins, the effective diffusion coefficient was found to be influenced by the pore size of DEAE resins, but independent on ligand density. The pore diffusion of proteins in the MMI resins was increased with the increase of ligand density and pore size, which is mainly due to stronger hydrophobic interaction between the resins and the proteins. The dynamic adsorption capacity Q10%could be calculated from the breakthrough curves. For DEAE resins, Q10%of BSA was high and generally increased linearly with the equilibrium adsorption capacity. However the maximum Q10%of bIgG was obtained at the ligand density around100μmol/g gel, which was lower than that in static adsorption process. This behavior could be explained as a result of decreasing accessible surface area for bIgG during the dynamic adsorption process. However, Q10%of BSA with MMI resin was low, but that of bIgG was depended significantly on ligand density and pore size of the resins. Under low operational flow rates, the resin with high ligand density and medium pore size (MMI-B-4FF-110) could efficiently adsorb bIgG(4) The adsorption isotherms of bIgG/BSA protein mixtures with the three MMI resins, MMI-B-3.5HF-50, MMI-B-4FF-110and MMI-B-6FF-110, were investigated. Competitive adsorption was found during the adsorption processes. The results showed that bIgG could be preferentially adsorbed and the performance of MMI-B-4FF-110was the best among the three resins. The qm of bIgG with MMI-B-4FF-110was93.51mg/g, while that of BSA was only43.69mg/g. It was found that the adsorption of BSA was influenced by the addition of bIgG. Due to the preferential adsorption of bIgG, the adsorption capacity of BSA with MMI resins exceeded the equilibrium adsorption capacity at the beginning of the adsorption, and then decreased to the equilibrium adsorption capacity. This effect may due to the strength difference of the hydrophobic interaction between the MMI resin and the two proteins.(5) The chromatographic separation processes were developed using MMI resins for IgG separation. The protein loading and elution conditions were optimized for bIgG/BSA mixtures. High purity of bIgG could be obtained when the protein was loaded at pH7.0and eluted at pH4.0. MMI resins with different ligand densities and pore sizes were investigated and MMI-B-4FF-110was found to have the best performance. Furthermore, the optimized conditions were used to purify bIgG from crude bovine serum with MMI-B-4FF-110. The result showed that after the pretreatment with ammonium sulfate precipitation, the purity of bIgG was close to95%and the yield was more than85%with only one-step HCIC separation using MMI-B-4FF-110.In summary, DEAE ion exchange chromatography and MMI hydrophobic charge induction chromatography were studied and compared as the representative for conventional chromatographic method and the novel separation technique, respectively. The influence of pore size and ligand density was explored. MMI resins were found to have preferential adsorption of IgG. By optimizing the separation conditions, these resins could be used for effective purification of antibodies. The results would be useful to design and optimize the resins and improve the the separation efficiency for antibody purification.