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

Pegah Saremirad, Jeffery A. Wood, Yan Zhang, Ajay K. Ray*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

4 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)147-155
Number of pages9
JournalJournal of chromatography A
Volume1370
DOIs
Publication statusPublished - 28 Nov 2014
Externally publishedYes

Keywords

  • L-Arginine
  • Mathematical modeling
  • Protein refolding
  • Size exclusion chromatography

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