Existing mathematical models developed to describe membrane affinity chromatography are unable to match the complete breakthrough curve when a single Langmuir adsorption isotherm is used, because important deviations from the observed behaviour are systematically encountered in the simulation of breakthrough broadening near saturation. The relevant information required to overcome that limitation has been obtained by considering simultaneously both loading and washing curves, thus evaluating the adsorption data at equilibrium and recognizing what are the appropriate adsorption mechanisms affecting the observed behavior. The analysis indicates that a bi-Langmuir binding kinetics is essential for a correct process description up to the saturation of the stationary phase, together with the use of the relevant transport phenomena already identified for the experimental system investigated. The input parameters used to generate the resulting simulations are evaluated from separate experiments, independent from the chromatographic process. Model calibration and validation is accomplished comparing model simulations with experimental data measured by feeding pure human immunoglubilin G (IgG) solutions as well as a cell culture supernatant containing human monoclonal IgG1 to B14-TRZ-Epoxy2 bio-mimetic affinity membranes. The simulations obtained are in good agreement with the experimental data over the entire adsorption and washing stages, and breakthrough tailing appears to be associated to the reversible binding sites of the bi-Langmuir mechanism. Remarkably, the model proposed is able to predict with good accuracy the purification of IgG from a complex mixture simply on the basis of the results obtained from pure IgG solutions.

Influence of protein adsorption kinetics on breakthrough broadening in membrane affinity chromatography

DIMARTINO, SIMONE;BOI, CRISTIANA;SARTI, GIULIO CESARE
2011

Abstract

Existing mathematical models developed to describe membrane affinity chromatography are unable to match the complete breakthrough curve when a single Langmuir adsorption isotherm is used, because important deviations from the observed behaviour are systematically encountered in the simulation of breakthrough broadening near saturation. The relevant information required to overcome that limitation has been obtained by considering simultaneously both loading and washing curves, thus evaluating the adsorption data at equilibrium and recognizing what are the appropriate adsorption mechanisms affecting the observed behavior. The analysis indicates that a bi-Langmuir binding kinetics is essential for a correct process description up to the saturation of the stationary phase, together with the use of the relevant transport phenomena already identified for the experimental system investigated. The input parameters used to generate the resulting simulations are evaluated from separate experiments, independent from the chromatographic process. Model calibration and validation is accomplished comparing model simulations with experimental data measured by feeding pure human immunoglubilin G (IgG) solutions as well as a cell culture supernatant containing human monoclonal IgG1 to B14-TRZ-Epoxy2 bio-mimetic affinity membranes. The simulations obtained are in good agreement with the experimental data over the entire adsorption and washing stages, and breakthrough tailing appears to be associated to the reversible binding sites of the bi-Langmuir mechanism. Remarkably, the model proposed is able to predict with good accuracy the purification of IgG from a complex mixture simply on the basis of the results obtained from pure IgG solutions.
JOURNAL OF CHROMATOGRAPHY A
S. Dimartino; C. Boi; G. C. Sarti;
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/106807
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