title: Oxidative protein refolding on size exclusion chromatography at high loading concentrations: fundamental studies and mathematical modeling.
abstract: Size exclusion chromatography has been demonstrated as an effective method for refolding a variety of proteins. However, to date process development mainly relies on laboratory experimentation of individual factors. A robust model is essential for high-throughput process screening and optimization of systems to provide higher productivity and refolding yield. In this work, a detailed kinetic scheme of oxidative refolding of a model protein (lysozyme) has been investigated to predict the refolding results in SEC. Non-reactive native, quenched and equilibrium studies were conducted to obtain the model parameters for the species formed during refolding of denatured/reduced lysozyme. The model was tested in various operating conditions, such as: protein loading concentration, injection volume, flow rate and composition of refolding buffer with and without the use of l-arginine additive. An apparent two-state mechanism was found adequate to describe refolding of lysozyme on SEC for the operating condition tested in this work. Furthermore, using low concentration of l-arginine combined with urea as common aggregation suppressor additives showed insignificant change in kinetics of refolding of lysozyme on SEC. However, addition of l-arginine changed mass transfer properties of some of the species formed in refolding reaction which was considered in the model to accurately predict the result of refolding on SEC.
authors.affiliation: Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Canada.
authors.forename: Pegah
authors.initials: P
authors.lastname: Saremirad
authors.affiliation: Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Canada.
authors.forename: Jeffery A
authors.initials: JA
authors.lastname: Wood
authors.affiliation: Faculty of Engineering & Applied Science, Memorial University of Newfoundland, St John's, Canada.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Canada. Electronic address: aray@eng.uwo.ca.
authors.forename: Ajay K
authors.initials: AK
authors.lastname: Ray
mesh_terms: D002850:Chromatography, Gel / Q000379:methods*; D007700:Kinetics; D008954:Models, Biological; D009113:Muramidase / Q000032:analysis* / Q000737:chemistry; D010084:Oxidation-Reduction; D058849:Protein Refolding
chemical_list: D009113:Muramidase
keywords: Mathematical modeling; Protein refolding; Size exclusion chromatography; l-Arginine