A novel two-zone protein uptake model for affinity chromatography and its application to the description of elution band profiles of proteins fused to a family 9 cellulose binding module affinity tag.
A novel two-zone model (TZM) is presented to describe the rate of solute uptake by the stationary phase of a sorption-type chromatography column. The TZM divides the porous stationary-phase particle into an inner protein-free core and an outer protein-containing zone where intraparticle transport is limited by pore diffusion and binding follows Langmuir theory. The TZM and the classic pore-diffusion model (PDM) of chromatography are applied to the prediction of stationary-phase uptake and elution bands within a cellulose-based affinity chromatography column designed to selectively purify proteins genetically labelled with a CBM9 (family 9 cellulose binding module) affinity tag. Under both linear and nonlinear loading conditions, the TZM closely matches rates of protein uptake within the stationary phase particles as measured by confocal laser scanning microscopy, while the PDM deviates from experiment in the linear-binding region. As a result, the TZM is shown to provide improved predictions of product breakthrough, including elution behavior from a bacterial lysate feed.
Kavoosi
Mojgan
M
Michael Smith Laboratories and the Department of Chemical and Biological Engineering, The University of British Columbia, Vancouver, BC, Canada V6T 1Z4.
Sanaie
Nooshafarin
N
Dismer
Florian
F
Hubbuch
Jürgen
J
Kilburn
Douglas G
DG
Haynes
Charles A
CA
D000327:Adsorption; D002482:Cellulose / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003911:Dextrans; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008956:Models, Chemical; D016062:Porosity; D011506:Proteins / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D020124:Thermotoga maritima; D013997:Time Factors
D003911:Dextrans; D011506:Proteins; D011993:Recombinant Fusion Proteins; D002482:Cellulose; C025614:sephadex