title: Microcalorimetric study of the adsorption of PEGylated lysozyme on a strong cation exchange resin.
abstract: Adsorption of native as well as mono-, di-, and tri-PEGylated lysozyme on Toyopearl Gigacap S-650M in sodium phosphate buffer is studied by isothermal titration calorimetry and by independent adsorption equilibrium measurements at pH 6 and 25°C. The production and separation of PEGylated lysozyme is discussed. Two different PEG sizes are used (5 kDa and 10 kDa) which leads to six different forms of PEGylated lysozyme which were systematically studied. The sodium chloride concentration is varied according to the elution conditions in the production process. The specific enthalpy of adsorption Δh(p)(ads) is determined from the calorimetric and the adsorption equilibrium data. It was found to be exothermal and constant with increasing adsorber loading for native lysozyme. For all PEGylated forms there is an influence of the adsorber loading on Δh(p)(ads) which is found to become more important with increasing PEGylation degree (total molecular weight of conjugated PEG). At low loadings the adsorption of all PEGylated lysozyme forms is exothermal. With increasing loading the adsorption becomes less exothermal and for the species with higher PEGylation degree also endothermal effects are observed at higher loadings. A thermodynamic analysis is carried out by which the enthalpic and entropic contributions to the binding constants are quantified. The findings are discussed on a molecular level. The results provide insight into the adsorption mechanisms of polymer-modified proteins on chromatographic resins.
authors.affiliation: Laboratory of Engineering Thermodynamics, University of Kaiserslautern, Kaiserslautern, Germany. tim.blaschke@mv.uni-kl.de
authors.forename: Tim
authors.initials: T
authors.lastname: Blaschke
authors.forename: Jefferson
authors.initials: J
authors.lastname: Varon
authors.forename: Albert
authors.initials: A
authors.lastname: Werner
authors.forename: Hans
authors.initials: H
authors.lastname: Hasse
mesh_terms: D000327:Adsorption; D002151:Calorimetry / Q000379:methods; D002411:Cation Exchange Resins / Q000737:chemistry*; D002412:Cations / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000295:instrumentation*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
chemical_list: D002411:Cation Exchange Resins; D002412:Cations; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D009113:Muramidase