Nonlinear immobilized metal affinity chromatography of proteins

The development of efficient bioseparation processes for the production of high purity biopharmaceutical products is one of the most pressing engineering challenges facing the pharmaceutical and biotechnology industry today. Although preparative immobilized metal affinity chromatography (IMAC) is increasingly being used for protein purification, the choice of operating conditions has remained largely empirical, resulting in sub-optimal performance of these separation systems. The magnitude with which key experimental variables influence the affinity and specificity of 'immobulized metal-protein' adsorption is unknown due to the absence of any systematic quantitative evaluations. Quantifying the influence of the operating variables on protein adsorption is crucial for the understanding of the chromatographic process, and in turn would facilitate the design of rapid and efficient large scale protein separations. In this dissertation a rigorous multicomponent adsorption formalism (MAIC) that considers the multipoint nature of protein adsorption, the possibility of steric hinderance of stationary phase sites upon binding of macromolecules, and the role of mobile phase modifier is presented. The MAIC equilibria in concert with the chromatographic mass transport equations is shown to accurately predict the experimental results in all modes of nonlinear chromatography. Numerical simulations are subsequently employed to carry out a detailed investigation of the influence of the operating variables on protein purification. These results provide significant insights on the separation process and heed in the development of efficient isocratic and gradient operations.; Theoretical predictions and experimental results indicate that the traditional mobile phase modifiers, that bind to the IDA-Cu(II) surface with a single interaction site displace proteins that bind with multiple interaction sites. The ability of these high affinity modulators to act as displacers has important implications in linear and step gradient chromatography. In IMAC step gradient chromatography, step changes in modifier concentrations do not travel at the linear velocity of the column but do so at a velocity characteristic of the adsorption equilibrium. The high affinity of these compounds presents the possibility of step gradient chromatography exhibiting displacement like behaviour, with isotachic protein zones and sharp protein-protein boundaries. Further, linear gradients in these systems would be subject to deformation due to the self sharpening nature of their adsorption equilibria, potentially resulting in displacement like behaviour.; Counter to traditional understanding, protein separations using high affinity modulators do not necessarily increase the developmental length for nonlinear separations. In gradient chromatography, the 'modulator-protein' interference effects lead to the spiking of the modulator between the feed components; thus acting as spacer displacers. Due to their ability to act as displacers these modulator spikes affect sharp protein rear; thereby decreasing the interface shock layer thickness and improving resolution. Similar improvement in resolution can also be obtained in elution chromatography by choosing an appropriate modulator concentration, and loading the feed mixture in the absence of any modulator. These results prompt the use of high affinity low molecular weight modulators to improve separations in other chromatographic systems as well.