Crosslinked glass fiber affinity membrane chromatography and its application to fibronectin separation
Introduction
Membrane-based affinity chromatography has been extensively used for biomolecule purification and immunoadsorption [1], [2], [3], [4], [5]. The main advantages of using synthetic membranes compared to conventional bead supports are their incompressibility and the fact that they eliminate internal diffusion limitations. Most investigations have focused on the preparation and modification of the supporting membranes, because, regarding affinity adsorption and elution, abundant information had already accumulated from classical column affinity chromatography [6]. Affinity membranes can be employed as flat sheets, monolithic discs [7] and hollow fibers [8], [9].
In membrane affinity chromatography, separation is based on the affinity characteristics of the solute molecules and not their size. For this reason, large pore sizes and high porosities should be used, in order to ensure that even the large impurities pass through. Most of the common commercially available membranes are not suitable for membrane affinity chromatography [10]. In our previous papers [11], [12], [13], [14], macroporous cellulose membranes were prepared from high quality filter paper by mercerization followed by chemical crosslinking. The prepared membranes possessed a high porosity (∼50%) and large pores (0.4–1.0 μm), had a low cost and were suitable because of their long durability, among other reasons, for affinity chromatography. The prepared membranes were further modified and activated by introducing epoxyl, triazine, aldehyde or diazonium groups, which were then coupled with enzymes or other affinity ligands. Using the prepared affinity membranes, the separation and purification of concanavalin A [11], trypsin inhibitors [12], serum proteins, globulins [14], papain inhibitors [13] and peroxidase [14] were carried out.
Besides the cellulose membranes mentioned above, we found that glass fibers are also suitable for the preparation of affinity membranes. In classical column affinity chromatography, the glass beads are the most important inorganic supporting material, because glass and its derivatives have high stability and are not attacked by microorganisms [15]. However, in membrane affinity chromatography, glass, like other inorganic materials, cannot be easily processed as a membrane because it is very brittle. In a previous paper [16], affinity membranes were prepared from commercial glass fiber filters. They were first treated with a piranha solution (a mixture of 70% vol. concentrated sulfuric acid (98%, m/m) and 30% vol. of a hydrogen peroxide solution (30%, m/m)) to enrich the number of silanol groups, followed by silanization to introduce epoxyl or amino moieties as functional groups. Affinity ligands were then immobilized using a glutaraldehyde, diazotization or carbodiimide pathway. The three immobilization methods were investigated in detail by determining and comparing the activities of the immobilized enzymes. The results indicated that the glutaraldehyde method provided the highest performance regarding enzyme immobilization and the highest durability, because of its crosslinking capability.
The objective of this paper was to prepare macroporous glass membranes from glass fiber filters using several chemical crosslinking procedures. The filters were first treated with a piranha solution, to clean the membrane and to increase the surface density of the OH groups. They were then crosslinked using one of the following crosslinkers: bis[3-(trimethoxysilyl)-propyl]amine, 1,2-bis(triethoxysilyl)-ethane or 1,3-diethoxy-1,1,3,3-tetramethyldisiloxane. After crosslinking, the membranes were modified by introducing NH2 or epoxyl functional groups. The epoxyl groups were thereafter converted to aniline moieties. Ligand immobilization was carried out via diazotization or using glutaraldehyde as a coupling reagent. Characterization of the membranes was carried out using bovine serum albumin and trypsin as test ligands. By using successively two cartridges containing gelatin and heparin immobilized affinity membranes, respectively, fibronectin from human blood plasma could be separated. The separations were monitored and evaluated by electrophoresis.
Section snippets
Chemicals
Sigma glass fiber filters (1.5-μm retention), trypsin (type IX), Nα-benzoyl-l-arginine p-nitroanilide (BAPNA), bovine serum albumin (BSA; 99%), human serum albumin (HSA; 99%), gelatin (type A), heparin (140 USP units per mg), fibronectin (FN), phenylmethylsulfonyl fluoride (99%) and human plasma were purchased from Sigma (St. Louis, MO, USA). Bis[3-(trimethoxysilyl)-propyl]amine (90%), 1,2-bis(triethoxysilyl)-ethane (96%), 1,3-diethoxy-1,1,3,3-tetramethyldisiloxane (97%),
Results and discussion
In our previous work [16], membrane affinity chromatography was carried out on silanized glass membranes, which were prepared from glass fiber filters. Several ligand immobilization pathways have been employed. Among them, the glutaraldehyde method provided the best results, both regarding membrane stability and ligand immobilization capacity. The higher stability occurred because the glutaraldehyde molecules condensed into larger molecules, which crosslinked the glass fibers (Fig. 1).
In the
Conclusion
Affinity membranes were prepared from glass fiber filters by first treating them with a piranha solution, followed by crosslinking. Of three bifunctional silanes used as crosslinkers, 1,2-bis(triethoxysilyl)-ethane was found to provide the most compact structure. The crosslinked membranes were further modified with either γ-aminopropyltriethoxysilane or γ-glycidoxypropyltrimethoxysilane to introduce amino and epoxyl groups, respectively. Affinity ligands, gelatin and heparin, were immobilized
Acknowledgements
This work was supported by the National Science Foundation.
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