Scale up of a chromatographic capture step for a clarified bacterial homogenate - Influence of mass transport limitation and competitive adsorption of impurities.

A model-based approach for scaling up chromatographic capture step was developed. The purification of human basic fibroblast growth factor protein 2 (FGF2) from an E. coli homogenate on a cation exchange resin was selected as a case study. Non-ideal effects accompanying the capture operation were examined, including: reduction in the protein diffusivity in the presence of the homogenate, competitive adsorption between FGF2 and undefined impurities, and flow behavior in external column volumes. The viscosity of the homogenate was measured as a function of dilution degree and shear stress, and its contribution to the diffusivity reduction was quantified. A dynamic model was formulated which accounted for underlying kinetic and thermodynamic dependencies. The model parameters were determined for a lab scale system using a small 2-mL column. The model was successfully used to scale up the capture operation from the lab scale column to a preparative bench scale column of about 1 L volume.

Effect of flow behavior in extra-column volumes on the retention pattern of proteins in a small column.

Experimental and theoretical analysis of deformation of band profiles in extra-column volumes (ECV) was performed, and its influence on the retention pattern of proteins in a small chromatographic column was quantified. Several macromolecule and small-molecule compounds, and their mixtures were eluted from a chromatographic system in the absence and presence of the column. The peak deformation in ECV was attributed to non-uniform velocity distribution in the radial direction in connecting capillaries. The phenomenon enhanced with increasing molecular weight of the model compound, when radial diffusion dominated the mechanism of band spreading. The band shape was also affected by the geometry of the injection system used, i.e., an injection loop capillary or a superloop. The phenomenon vanished for a small molecule compound, for which plug flow conditions could be established. The difference in flow behaviour of the macromolecule and small-molecule compounds caused them to migrate with different velocities in ECV, which resulted in partial separation of their bands. The ECV effect influenced the retention behaviour of macromolecules in a small column; it caused tailing of peaks and asymmetry of breakthrough curves. To describe the elution profiles in ECV and in the column, a mathematical model was used which accounted for non-ideality of the flow pattern. The model reproduced accurately band profiles of macromolecules within a range of relatively low velocities, typical however for protein chromatography.

Retention Behavior of Polyethylene Glycol and Its Influence on Protein Elution on Hydrophobic Interaction Chromatography Media.

The retention behavior of polyethylene glycol (PEG) on different types of hydrophobic interaction chromatography (HIC) resins containing butyl, octyl, and phenyl ligands was analyzed. An incomplete elution or splitting of the polymer peak into two parts was observed, where the first one was eluted at the dead time of the column, whereas the second one was strongly retained. The phenomenon was attributed to conformation changes of the polymer upon its adsorption on hydrophobic surface. The effect enhanced with increasing molecular weight of the polymer and hydrophobicity of the HIC media. Addition of PEG to the mobile phase reduced binding of proteins to HIC resins, which was demonstrated with two model systems: lysozyme (LYZ) and immunoglobulin G (IgG), and their mixtures. In case of LYZ, the presence of PEG caused reduction in the protein retention, whereas for IgG-a decrease in efficiency of the protein capture. The effect depended on the adsorption pattern of PEG; it was pronounced in the systems in which conformational changes of the polymer were suggested to occur.

Prediction tool for loading, isocratic elution, gradient elution and scaling up of ion exchange chromatography of proteins.

An efficient mathematical tool for the design and scaling up of protein chromatography is suggested, in which the model parameters can be determined quickly over a wide operating space without large material investments. The design method is based on mathematical modelling of column dynamics and moment analysis. The accuracy of the dynamic models that are most frequently used for simulations of chromatographic processes is analyzed, and possible errors that can be generated using the moment analysis are indicated. The so-called transport dispersive model was eventually employed for the process simulations. The model was modified to account for the protein dispersion in void volumes of chromatographic systems. The manner of the model calibration was suggested, which was based on a few chromatographic runs and verified over a wide space of the operating parameters, including composition and flow rate of the mobile phase, column dimensions, residence time, and mass loading. The model system for the study was ion-exchange chromatography. The analysis was performed based on the elution profiles of basic fibroblast growth factor 2 and lysozyme, on two different IEX media.