New Mixed-mode Adsorbents And The Application For The Separation Of Bovine Colostrum Immunoglobulin

Mixed-mode chromatography is a new type of biological separation technology, which possesses functional ligands with various interaction modes, leading to high adsorption capacity, high selectivity and mild elution. In this thesis, new mixed-mode adsorbents were screened and prepared to isolate and purify bovine colostrum immunoglobulin, by integrating the mechanism of thiophilic chromatography and hydrophobic charge-induction chromatography. Thiophilic chromatography has shown a selective binding for immunoglobulin in the presence of high salt concentration, while the typical salt-independent properties were found by hydrophobic charge induction chromatography. Combining the advantages of thiophilic, hydrophobic and electrostatic interactions, mixed-mode chromatography could provide an effective adsorption under both high and low salt concentrations. The adsorption behavior is significantly influenced by pH, making a convenient elution. In this thesis, three main aspects were focused, including adsorbents preparation, adsorption properties and application for the separation of bovine colostrum immunoglobulin.With the agarose gel Bestarose4FF as the matrix and divinylsulfone as the activation reagent, a series of mixed-mode adsorbents were prepared with the functional ligands of4-mercaptoethylpyridine,2-mercaptoimidazole,2-mercapto-l-methylimidazole and3-mercapto-1,2,4-triazole. New adsorbents prepared were named as Bestarose-DVS-MEP, Bestarose-DVS-M1, Bestarose-DVS-MMI and Bestarose-DVS-MTI, respectively. The activation and coupling conditions were optimized, resulting in the ligand density of60～70μmol/ml and the coupling efficiency of60%.The adsorption isotherms and kinetics of new adsorbents for bovine colostrum immunoglobulin were studied. The results showed that Bestarose-DVS-MEP, Bestarose-DVS-MI, Bestarose-DVS-MMI and Bestarose-DVS-MTI had high capacities and showed salt-independent properties. Meanwhile, sulfate addition could accelerate the adsorption with less influence on the capacities. The adsorption behaviors of Bestarose-DVS-MEP, Bestarose-DVS-MI and Bestarose-DVS-MMI were sensitive to pH. Hence, the efficient adsorption and elution could be achieved at pH7and pH4, respectively.Finally, new mixed-mode adsorbents including Bestarose-DVS-MEP, Bestarose-DVS-MI, Bestarose-DVS-MMI and Bestarose-DVS-MTI were applied to purify immunoglobulin from bovine colostrum powder solution. After comparison, Bestarose-DVS-MI was chosen as the best one, with high adsorption, good pH sensibility and high selectivity for bovine colostrum immunoglobulin. Moreover, the chromatographic conditions were optimized. The optimized conditions were obtained by loading1ml sample with25mg/ml initial concentration at pH7and elution at pH4. The purity reached above95%, the purification factor was about6.2, and the recovery of immunoglobulin was about64.2%.In the present work,2-mercaptoimidazole was chosen as a suitable ligand of mixed-mode adsorbent for separation of immunoglobulin from bovine colostrum powder solution. This technology may lead a new way to utilize the bovine colostrum for multiple products with high additional value.