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  • Oxidative protein refolding on size exclusion chromatography at high loading concentrations: fundamental studies and mathematical modeling.

Oxidative protein refolding on size exclusion chromatography at high loading concentrations: fundamental studies and mathematical modeling.

Journal of chromatography. A (2014-12-03)
Pegah Saremirad, Jeffery A Wood, Yan Zhang, Ajay K Ray
RESUMEN

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.

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