Modeling chromatographic purification of rh-bFGF with Heparin HyperD affinity sorbent using a homogeneous and a pore diffusion model

Abstract

On the basis of the purification of recombinant human basic fibroblast growth factor (rh-bFGF), the prediction of exact protein adsorption and gradient elution by two mathematical models was investigated. To this end, thermodynamic and kinetic parameters were employed from batch experiments. These were evaluated from both the film and pore diffusion (FPD) and the homogeneous diffusion model, and applied to a packed bed. The adsorption of rh-bFGF as a function of the salt concentration on Heparin HyperD was well described by the Langmuir model. It was possible to adopt the functional connection between ionic strength and dissociation constant as well as maximal adsorption capacity for the calculation of elution processes using the FPD model. Uptake curves displayed a significant increase in the effective pore diffusion coefficient, D _p, with decreasing protein concentration, which is indicative for surface diffusion. In contrast, the coefficient for homogeneous diffusion, D _h, was independent of the protein concentration. Adaptation to the packed bed required a substantial increase in both D _p and D _h. Although this could be seen as an indication of convective flow in the sorbent, data from different and relatively slow mobile phase velocities clearly indicated absence of intra particulate flow. For an exact prediction of the adsorption process over the wide range of concentrations applied here, a still deeper insight into protein adsorption and desorption is needed to discriminate among the various diffusion and adsorption phenomena.

Acknowledgments

We thank A. Walter for the technical assistance.