Predicting the elution behavior of proteins in affinity chromatography on non-porous particles

Authors
Citation
Wc. Lee et Ch. Chen, Predicting the elution behavior of proteins in affinity chromatography on non-porous particles, J BIOCH BIO, 49(1-3), 2001, pp. 63-82
Citations number
30
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS
ISSN journal
0165-022X → ACNP
Volume
49
Issue
1-3
Year of publication
2001
Pages
63 - 82
Database
ISI
SICI code
0165-022X(20011030)49:1-3<63:PTEBOP>2.0.ZU;2-#
Abstract
Affinity chromatography on non-porous particles of microsize is particularl y useful for the rapid analysis and micropreparative separation of proteins . The elution behavior of proteins in an affinity column packed with non-po rous copolymerized particles of styrene, methyl methacrylate and glycidyl m ethacrylate was investigated both theoretically and experimentally, using t he lysozyme-Cibacron Blue 3G-A affinity system. Equations used to predict t he elution profiles, resulting from the elution by increasing the ionic str ength (NaCl concentration) in the mobile phase, were obtained. The maximum adsorbate concentration, desorption rate constant and equilibrium constant under elution conditions were determined by matching experimental data with predicted elution profiles. Based on the parameters determined at a flow-r ate of 0.5 ml/min and with 1 M NaCl in the elution buffer, the model equati ons could predict the elution profiles for other experimental runs, where d ifferent flow-rates and sodium chloride concentrations were used. Both the experimental and predicted results revealed that the affinity interaction k inetics are not significantly influenced by the flow-rate and, hence, the f ilm mass transfer. To elute bound lysozyme from immobilized dye ligand, a h igher value of the ionic strength leads to a faster elution and a sharper e lution peak. The influence of elution conditions on the kinetic and thermod ynamic parameters and, consequently, on the elution peak profiles was evalu ated. The model equations can also predict the behavior of protein elution from an affinity column by changing the pH of the mobile phase, according t o a previous study. (C) 2001 Elsevier Science B.V. All rights reserved.