Application of dye affinity membrane chromatography to protein purification from raw materials. Comparison with soft-gel matrices.

F. J. WOLMAN; M. GRASSELLI; O. CASCONE

Handook of Membrane Research: Properties, Performance and Applications

Stephan V. Gorley, Nova Science Publishers, Inc

Lugar: Hauppauge, NY, USA; Año: 2010; p. 367 - 385

Abstract The usefulness of affinity membrane chromatography for protein purification from raw materials was assessed by comparison of the performance of membranes with a triazinic dye as the affinity ligand with that of soft-gels with the same ligand attached. A neutral protease from a commercial powder preparation and lactoferrin (Lf) from bovine whey and colostrum were used as the model proteins to be purified from complex samples. High-capacity dye-affinity hollow fibres were prepared by radiation-induced grafting of methacrylic and acrylamide monomers onto microfiltration polysulfone membranes. This adsorptive grafted layer can be modified in their hydrophilicity by changing the monomer composition. The hydrophilicity degree brought about an important effect on ligand density immobilisation and also on protein adsorption capacity. The triazinic dyes Yellow HE-4R and Red HE-3B were immobilised and used as affinity ligands for purification of a neutral protease and Lf respectively. In both cases, the more hydrophilic adsorptive membranes showed the best chromatographic properties.             For neutral protease purification from a commercial preparation, a maximum capacity of 24220 U/ml membrane and a dissociation constant of 91 U/ml was calculated from the corresponding isotherm. Elution with 2 M NaCl at pH 4 allowed quantitative recovery of the adsorbed protease. A yield of 72% with a purification factor of 3.7 was achieved. Productivity was six times higher than that attained with the same dye attached to a soft gel. The adsorptive capacity of Red HE-3B membranes to Lf solution were twelve times higher than that obtained with the same ligand immobilised on agarose beads. Using colostrum as the Lf source in a batch system, more than 90 % of the Lf contained in that raw material was adsorbed by the dye-membranes while only 56 % was adsorbed by the dye-agarose beads. In the elution step, membranes reach 99% recovery versus 80% of agarose beads using 2 M NaCl in 25 % ethylene glycol. Final productivity for a cartridge assembled with Red HE-3B hollow fibres was 500 % higher than that attained with the dye agarose matrix.             In both cases, the adsorption step was nearly independent on the flow rate. In the elution step it was necessary to decrease the flow rate in order to maximise the product recovery. As a consequence of this non-convective behaviour in the elution step, membrane whole productivity was lesser than predicted though far higher than that of soft gels.