Research On Preparation And Application Of Rigid Ceramic/Agarose Composite Adsorbents For Protein Purification

Liquid chromatography, as an important unit in bioseparation process, is the most effective and widely used technology with the advantages of high resolution, simple operating equipment and high bioactivity preservation. The chromatographic media is the key of this technology. With the advance of technology and the increase of requirements for biological products, the performances of chromatographic packing materials have been continuously improved. The present work was aimed at developing a new chromatographic media with novel structure, mostly focused at preparation, functionalization and application of rigid ceramic/agarose composite adsorbents for liquid chromatography. The main works were carried out as follows:(1) Preparation of porous silica beads. Firstly, the ceramic pulp was fabricated by silica powders as skeleton, PEG10000 as porogenic agent and sodium alginate solution as binder. Later, the ceramic pulp was prepared into elemental silica particles by the method of oil emulsion and immobilized by calcium chloride. Then, the final porous silica beads with high porosity, strong bonds and good sphericity were obtained through high temperature sintering. Nitrogen adsorption-desorption analysis given a BET surface area of 28.75 m2/g, a BJH mean pore size of 33.3 nm and a porosity of 0.246 m3/g, respectively. The ceramic beads had good hydromechanics with the back pressure of 0.18 MPa at the flow velocity of 770 cm/h.(2) Preparation of porous ceramic/agarose composite beads. The porous of ceramic beads were filled with agarose solution. After mildly grinding with water in a mortar, the gel outside of the beads was removed by screening with standard sieve and elutriation in a fluidized bed. The composite particles had a wet density of 1.70 g/mL and a mean size of 65.3μm. The composite beads were superior to the traditional agarose based beads of 6FF in hydromechanics, column efficiency and column packing by the performance evaluation. Above all, it was appropriate for preparative chromatography.(3) Functionalization of the composite beads as cation ion-exchanger. Firstly, the beads were double cross-linked with glycol diglycidyl ether and epichlorohydrin. Then, carboxymethyl (CM) groups were coupled to the composite beads and the total ionic capacity reaches 72.4μmol/mL. Finally, The adsorption isothermal, adsorption dynamics, breakthrough curves were determined by using lysozyme as model protein. The saturated adsorption capacity was 55.9 mg/mL. The adsorption dynamics dates were well fitted with the pseudo second-order kinetic mode, and the composite beads could be operated at high velocity without loosing binding capacity.(4) Functionalization of the composite beads as GSH-adsorbents and purification of GST-ADAM15. Firstly, The BDGE activation efficiency and GSH ligand density reached 13.1 and 10.2μmol/mL, respectively. And the final coupling efficiency reached 77.8 %. Then, the chromatographic process of the GSH-adsorbents as function of elution mode, sample injection and operating velocity were evaluated. The results indicate that the GSH affinity adsorbents were provided with high specifity. The target protein could be adsorbed completely and target purity could be obtained with both of linear gradient and 100 % B solution elution. The composite adsorbents prepared were suitable for purification of fusion protein at high velocity.