Alternatives to conventional packed-bed capture and purification of monoclonal antibodies has been the motivation of many studies over the past decade. The work presented here fully characterizes the transport phenomena, affinity-adsorption, and elution of IgG in a commercial convective interaction media (CIM) Protein A monolithic column. The moment analysis method was employed to determine mass transfer parameters, namely axial dispersion, and evaluate the column efficiency through the height equivalent of a theoretical plate (HETP) using several tracer molecules covering a large range of molecular weights. The frontal analysis approach in the staircase mode was successfully applied for the first time in monolithic media to determine the dynamic binding isotherm for human IgG. In addition, elution was performed over a range of operating conditions to determine the critical pH needed to initiate desorption and subsequently the effects of pH and flow rate on the recovery of the bound IgG.
A complete chromatographic model was used to fit the data for each section of the separation process. The chromatographic model accounts for all mass transport and intrinsic rate parameters of the solute including axial convection, longitudinal dispersion, adsorption/desorption at the surface of the solid phase, and the extra-column effects associated with the mixing volumes and delay times of the circuit. The model was used to simultaneously fit the IgG effluent data for all of the operating conditions investigated in order to determine the kinetic parameters associated with breakthrough, washing, and elution steps. In particular, a detailed study was performed to investigate different kinetic models and the effect of pH on the elution kinetic parameters. The model showed a good agreement with the experimental data, elucidating both the predictive capability of the model and the reliability of the experimentally determined mass transfer parameters.
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