3D structure-based protein retention prediction for ion-exchange chromatography.

The interest in understanding fundamental mechanisms underlying chromatography drastically increased over the past decades resulting in a whole variety of mostly semi-empirical models describing protein retention. Experimental data about the molecular adsorption mechanisms of lysozyme on different chromatographic ion-exchange materials were used to develop a mechanistical model for the adsorption of lysozyme onto a SP Sepharose FF surface based on molecular dynamic simulations (temperature controlled NVT simulations) with the Amber software package using a force-field based approach with a continuum solvent model. The ligand spacing of the adsorbent surface was varied between 10 and 20A. With a 10A spacing it was possible to predict the elution order of lysozyme at different pH and to confirm in silico the pH-dependent orientation of lysozyme towards the surface that was reported earlier. The energies of adsorption at different pH values were correlated with isocratic and linear gradient elution experiments and this correlation was used to predict the retention volume of ribonuclease A in the same experimental setup only based on its 3D structure properties. The study presents a strong indication for the validity of the assumption, that the ligand density of the surface is one of the key parameters with regard to the selectivity of the adsorbent, suggesting that a high ligand density leads to a specific interaction with certain binding sites on the protein surface, while at low ligand densities the net charge of the protein is more important than the actual charge distribution.