Functionalized cellulose monolith based affinity chromatography columns for efficient separation of protein molecules

Abstract

Cellulose monoliths are prepared from cellulose acetate (CA) monoliths by the thermally induced phase separation technique followed by hydrolysis of the acetyl groups of the side chain of CA in a basic solution of sodium hydroxide. The morphology of the cellulose monoliths is found to be dependent on the fabrication conditions such as the concentration of CA, solvent ratio and cooling temperature. A comparison with ethylene-vinyl alcohol copolymer (EVOH) and poly(methyl methacrylate) (PMMA) monoliths reveals that cellulose is a better column material in terms of strength, liquid permeability and suppression of non-specific adsorption. To design the affinity columns for selective biomolecule adsorption and separation, N-hydroxysuccinimide (NHS) esters are immobilized over the cellulose monoliths by the N,N′-disuccinimidyl carbonate (DSC) activation method in the presence of N,N-dimethyl-4-aminopyridine (DMAP). The activated cellulose monoliths are allowed to react with Protein A under optimum conditions at the NHS ester sites to obtain affinity chromatography carriers for selective adsorption of Immunoglobulin G (IgG) protein by employing the association of Protein A with the IgG antibody. The adsorption capacity and adsorption efficiency are found to improve with the activated cellulose monoliths. It is revealed that cellulose monolith exhibited unique surface morphology with deep roughness in the pore structure, contributing to the enhancement of adsorption capacity. The desorption characteristics of IgG at different flow rates are found to be almost the same demonstrating that the elution time can be shortened by using higher flow rates. The high permeability coefficient of the monolith column promotes rapid diffusion to increase the flow velocity. The columns can be regenerated and reused without appreciable loss of adsorption efficiency. Such high efficiency separation of protein molecules cannot be achieved with traditional and commercially available agarose gel beads.