Fabrication Of New Cryogel Materials For Protein Chromatography

Cryogel monoliths have been extensively studied in chromatography ofbiological substances as macroporous continuous bed materials. However, cryogelbed often suffers from the low adsorption capacity due to its low specific surface area.The thesis focuses on the development of two methods for solving the problem.Moreover, cryogel monolith was extended to the use in steric exclusionchromatography (SXC).A “post-embedding modification” method was developed to enhance the specificsurface area of cryogel materials. Polymeric resin particles were incorporated insupport matrix, which result in rough pore wall and consequently twofold increase ofspecific surface area. The composite cryogel with ion-exchange polymer tentaclesacquired higher binding capacity for bovine serum albumin (BSA),6mg/mL, whichis2.8folds of normal cryogel. The capacity and column efficiency of cryogel bedcolumn changed slightly in a wide flow rate range (0.5-20cm/min). The compositecryogel can be used for fast primary separation of plasmid DNA from crude celllysate.A “double modification” method was designed to improve ion-exchange capacityof cryogel materials. Branched polyethylenimine was grafted on the support firstlyand diethylaminoethylchloride was grafted sequentially. The maximum capacity ofcomposite cryogel for BSA reached11.2mg/mL. The ion-exchange cryogel bed ofhigh capacity was practical for rapid protein chromatography.SXC is a new mode of protein chromatography, in which large proteins areretained on hydrophilic stationary phase surface due to the steric exclusion ofpolyethylene glycol (PEG) in the mobile phase, and the retained proteins can beeluted by reducing PEG concentration. The cryogel monoliths with interconnectedpores (10-100μm) allow much easy flow-though of viscous PEG buffer, so the SXCcan be operated at low back pressure. The dynamic binding capacity of the cryogelmonolith for γ-globulin was20mg/mL bed volume, much higher than those ofcryogel beds in adsorption-based chromatography. Globulin precipitates on the gelsurface was observed by scanning electron microscope. In the separation of bovineserum proteins, albumin was recovered in the breakthrough fraction with high purity,and globulin was over eight times concentrated in the elution pool. Rapid serum protein separation and concentration was demonstrated by SXC on the cryogelmonolith.Three kinds of monomers, viz. ionic liquid monomer (vinyl-butylimidazoliumchloride), zwitterionic monomer (carboxybetaine methacrylate) and BSA, wereresearched for fabrication of new cryogel monoliths respectively. Microscopeobservation was analyzed to evaluated the structure of cryogel matrix. BSA-basedcryogel was a prior chromatographic materials comparing with the other two.