Surface Modification Of Cryogel Monoliths For Protein Chromatography

Cryogel monoliths with interconnected macropores(10–100 ?m) and spongy structure have been extensively employed for the separation and purification of target biologics such as proteins and viruses directly from crude feedstocks. However, cryogel bed often suffers from the low adsorption capacity due to its low specific surface area. In order to enhance the protein chromatographic performance of cryogel, two kinds of novel cryogel monoliths with different surface structures were prepared by surface modification in the thesis for ion-exchange chromatography and steric exclusion chromatography.The cryogel surface was first modified by nanoparticles(NPs)-coating and subsequent double-modification for enhanced ion-exchange capacity of protein. It was found out that NPs-coating did not result in distinct changes of the column porosity and permeability. The rough pore wall surface and extended polymer chains offered more binding sites, so the dynamic binding capacity of the composite cryogel bed for bovine serum albumin reached 11.7 mg/mL bed volume at a flow rate of 6 cm/min, which was 4.2 times higher than that of the single-modified cryogel bed. The binding capacity as well as column efficiency decreased only slightly with increasing flow rate from 0.6 to 12 cm/min. The results indicated that the strategy of NPs-coating incorporating with double ion-exchanger modifications is promising for enhancing cryogel capacities, and the novel material would be useful for high-speed protein chromatography.Moreover, the thesis proposes that grafting zwitterionic polymer onto cryogel surface enhances protein retention in steric exclusion chromatography(SXC) on cryogel monolith. SXC is based on the principle that the exclusion of polyethylene glycol(PEG) on both a hydrophilic chromatography surface and a protein favors their association, leading to the protein retention on the chromatography surface. Elution of the retained protein can be achieved by reducing PEG concentration. In this study, the surface of polyacrylamide-based cryogel monolith was modified by grafting zwitterionic poly(carboxybetaine methacrylate)(pCBMA), leading the increase in the surface hydrophilicity. pCBMA grafting did not result in the changes of the physical properties of the monolith column, and the columns maintained good recyclability in SXC. The effect of the surface grafting on the SXC behavior of ?-globulin was investigated in a wide flow rate range(0.6–12 cm/min). It was found that the dynamic retention capacity increased 1.4–1.8 times by the zwitterionic polymer grafting in the flow rate range of 1.5–12 cm/min. The mechanism of enhanced protein retention on the zwitterionic polymer-grafted surface was proposed.