title: Influence of protein and stationary phase properties on protein-matrix-interaction in cation exchange chromatography.
abstract: A large number of different stationary phases for ion-exchange chromatography from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (lysozyme, cytochrome c and two monoclonal antibodies) were determined for nine commercially available cation-exchange adsorbents. The linear gradient elution model in combination with a thermodynamic approach was used to analyse the characteristic parameters of the protein-stationary phase-interactions. Based on the pH dependency of the characteristic charge and the equilibrium constant for binding the differences between the standard Gibbs energies in the adsorbed and the solute state for the protein ΔG(P)° and the salt ΔG(S)° were calculated. The characteristic charge B of the proteins strongly depends on the molecular mass of the protein. For small proteins like lysozyme there is almost no influence of the stationary phase chemistry on B, while for the Mabs the surface modification strongly influences the B value. Surface extenders or tentacles usually increase the B values. The variation of the characteristic charge of the MABs is more pronounced the lower the pH value of the mobile phase is, i.e. the higher the negative net charge of the protein is. The standard Gibbs energy changes for the proteins ΔG(P)° are higher for the Mabs compared to lysozyme and more strongly depend on the stationary phase properties. Surface modified resins usually show higher ΔG(P)° and higher B values. A correlation between ΔG(P)° and B is not observed, indicating that non-electrostatic interactions as well as entropic factors are important for ΔG(P)° while for the B values the accessibility of binding sites on the protein surface is most important.
authors.affiliation: Institute for Biochemistry, University of Applied Sciences Mannheim, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Urmann
authors.forename: Mathias
authors.initials: M
authors.lastname: Hafner
authors.forename: Christian
authors.initials: C
authors.lastname: Frech
mesh_terms: D000327:Adsorption; D000906:Antibodies / Q000737:chemistry / Q000302:isolation & purification*; D002411:Cation Exchange Resins / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D045304:Cytochromes c / Q000737:chemistry / Q000302:isolation & purification*; D009113:Muramidase / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding
chemical_list: D000906:Antibodies; D002411:Cation Exchange Resins; D045304:Cytochromes c; D009113:Muramidase