Chitin and chitosan-based membrane chromatography for protein purification

Membrane chromatography is a promising process for the large-scale protein separation and purification. Because of the convective flow of protein solutions through the pores of microporous membranes, membrane chromatography allows a high flow rate at a low pressure drop, thus has a high productivity. Novel macroporous chitin and chitosan membranes with controlled pore sizes and porosity, good mechanical properties and attractive surface characteristics were prepared, and then employed for protein purification via various affinity ligands.; Macroporous chitosan membranes were prepared by (1) casting an acidic chitosan solution that contains silica particles of selected sizes; (2) removing the solvent by evaporation; and (3) dissolving the silica particles in an alkaline solution. Macroporous chitin membranes could be easily obtained by acetylating the chitosan membranes with acetic anhydride in methanol. Cross-linked chitosan membranes and chitin membranes are insoluble both in acidic and basic solutions. Because of their macroporous structures, good mechanical strength, the presence of numerous reactive groups (NH₂ and CH₂OH), as well as hydrophilic surfaces, these membranes are excellent candidates as adsorptive membranes and biomedical materials.; Due to the presence of numerous N-acetyl-D-glucosamine (GIcNac) moieties, the affinity ligands for lysozyme and wheat germ agglutinin, in chitin molecules, macroporous chitin membranes can be used, without further modification, for the affinity separation of lysozyme or wheat germ agglutinin. Due to their macroporous structures and high adsorption surface areas, chitin membranes have a large number of accessible binding sites, and thus have high adsorption capacity for lysozyme (∼50 mg/ml membrane) and wheat germ agglutinin (180 mg/g membrane). The membranes could separate high purity lysozyme from egg white or high purity wheat germ agglutinin from wheat germ.; p-Aminobenzamidine (PAB), a trypsin inhibitor, was attached to cross-linked chitosan membranes via succinic acid as spacers. PAB-chitosan membranes (2.7 × 10−3 mol PAB/g membrane) could purify trypsin from crude trypsin solutions, with a purification factor >7, a specific activity >9500 BAEE units/mg and activity yields of 60 to 80%. Various pseudospecific ligands (Cibacron Blue F3GA, Procion Red HE-3B and Procion Blue MX-R) for kinases, dehydrogenase and albumins were immobilized on macroporous chitosan and chitin membranes (10 ∼ 200 Umol dye/ml membrane). Cibacron Blue F3GA-chitosan membranes could separate high purity human serum albumin (8.4mg/ml membrane) from human plasma. Due to the presence of numerous NH₂ groups, cross-linked macroporous chitosan membranes (0.83meq/g membrane) can be employed for protein separations in the ion exchange mode. Highly selective and reproducible separations of ovalbumin, albumin or soybean trypsin inhibitor from mixtures of ovalbumin-lysozyme, human serum albumin-cytochrome C, or soybean trypsin inhibitor-cytochrome C could be achieved at pH 6.