Development of a low-cost pseudobioaffinity chromatographic matrix for lactoferrin purification from milk or whey

F. J. WOLMAN; A. A. NAVARRO DEL CAÑIZO; D. GONZÁLEZ MAGLIO; O. CASCONE

Barcelona

Congreso; 14th European Congress on Biotechnology; 2009

European Federation of Biotechnology

The main challenge of downstream processing of proteins is to have chromatographic matrices that optimise the performance/cost ratio, and pseudobioaffinity matrices can fulfil this condition by attachment of triazinic dyes to chemically cross-linked low-cost natural biopolymers.Bovine lactoferrin (Lf) was taken as the model as it is a natural antimicrobial protein of industrial interest present in milk and whey.Macrobeads of cross-linked chitosan were sinthesised by dissolving chitosan at a concentration of 2 % in 2 % acetic acid and dropping the solution on 1 M NaOH. Then the macrobeads (2 mm diameter) were cross-linked and derivatised with the dye Red HE-3B. Two cross-link reagents at concentrations of 50 mM and 100 mM were assayed: epichlorhidrin (E) and glutaraldehyde (G). Maximum capacities for Lf adsorption were 21.4 and 20.6 mg/g for E and G cross-linked beads respectively, and dissociation constants were 0.81 and 0.51 mg/g respectively. Different eluents at pH 7, 8 and 9 were assayed: 0.5 M NaSCN, 2 M NaCl, 25% ethyleneglycol, 0.5 M NaSCN in 25% ethyleneglycol and 2 M NaCl in 25% ethyleneglycol. Lf elution was maximal with 2 M NaCl in 25 % ethyleneglycol at pH 7 for both systems, being higher for E cross-linked beads (85 % vs 25 %). This result evidences that both electrostatic and hydrophobic interactions are involved in the Lf adsorption to the matrix. At pH 8 and 9 results were similar.For Lf purification from bovine colostrum, E cross-linked beads showed a better performance: at the same sample:matrix ratio, they adsorbed all the lactoferrin contained in the sample while G cross-linked beads only adsorbed 60 % of the Lf contained in the sample. The Lf recovery was also higher for the E cross-linked beads with an 86% of elution while only a 27% was eluted from the G cross-linked beads.To assess the suitability of E cross-linked beads for repetitive utilisation, five Lf purification batch processes were performed without significant loss in beads capacity.