title: Optimization of a chromatographic stationary phase based on gellan gum using central composite design.
abstract: To develop a new stationary phase of easy production, low cost, biocompatible, biodegradable and low unspecific adsorption, a three-dimensional network was prepared by combining the natural polysaccharide of gellan with divalent cations. The stability of this cation exchange chromatographic matrix was optimized by using an experimental design tool. The optimal conditions proposed for the gellan gel formulation were 48mM ZnSO4, 0% DMF, 25°C, 0.75% gellan and 0.5h. The applicability of gellan matrix was tested by chromatographic assays with three model proteins (bovine serum albumin (BSA), α-chymotripsin and lysozyme). The results showed that the retention occurred in function of the net charge of each protein in MES buffer pH 6.2 and the elution was performed by increase of ionic strength to 750mM NaCl in MES buffer pH 6.2. Lysozyme was the more retained protein due to its positive charge more effective than α-chymotripsin, while BSA did not interact with the matrix due to its negative charge at these conditions. Dynamic binding capacity assays were accomplished to characterize this matrix and to compare with commercial resins. The values of dynamic binding capacity from gellan gel were 3.9mg/mL and 17.4mg/mL, at 10% and 50% of breakthrough, respectively. In this way, gellan gel might be a promising chromatographic matrix to explore ionic interactions and to be applied in different purification strategies, getting the best benefit from its use at low cost.
authors.affiliation: CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-506 Covilhã, Portugal.
authors.forename: A I C
authors.initials: AI
authors.lastname: Gonçalves
authors.affiliation: CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-506 Covilhã, Portugal.
authors.forename: L A
authors.initials: LA
authors.lastname: Rocha
authors.affiliation: University of Cambridge, Cambridge System Biology Centre, Sanger Building, 80 Tennis Court Road, Cambridge CB2 1GA, England, United Kingdom.
authors.forename: J M L
authors.initials: JM
authors.lastname: Dias
authors.affiliation: CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-506 Covilhã, Portugal. Electronic address: lpassarinha@fcsaude.ubi.pt.
authors.forename: L A
authors.initials: LA
authors.lastname: Passarinha
authors.affiliation: CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-506 Covilhã, Portugal.
authors.forename: A
authors.initials: A
authors.lastname: Sousa
mesh_terms: D002852:Chromatography, Ion Exchange / Q000295:instrumentation* / Q000379:methods; D008956:Models, Chemical; D016571:Neural Networks, Computer; D011135:Polysaccharides, Bacterial / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D011135:Polysaccharides, Bacterial; D011506:Proteins; C048288:gellan gum
keywords: Central composite design; Chromatography; Gellan gum; Ion exchange; Matrix