Visualizing patterns of protein uptake to porous media using confocal scanning laser microscopy

Abstract

Confocal scanning laser microscopy has been used to visualize the uptake of fluorescence-labeled proteins to porous stationary phases in finite bath adsorption experiments. Reference proteins were labeled with three different fluorescent dyes and a porous cation exchanger was sequentially incubated with solutions of these protein–dye conjugates. This sequential incubation experiment was used to investigate the pattern of protein uptake during adsorption. The confocal images obtained during the experiments clearly visualized that under certain conditions the adsorbent particles are gradually saturated from the rim to the core, a pattern consistent with the shrinking core model. Changes in the mobile phase conditions (ionic strength, pH) can lead to significant shifts in the uptake pattern, i.e., the further transport of initially bound molecules to the core, thus making binding sites at the rim available for adsorption of new molecules. At low pH and ionic strength BSA showed an uptake pattern, which was completely in accordance with the shrinking core model, but a clear deviation from this profile was observed at increased ionic strength. During adsorption of a monoclonal antibody a change in pH was sufficient to change the uptake pattern in finite bath adsorption completely. An attempt was made to correlate this behavior to the shape of the equilibrium-binding isotherm. The technique presented here allows unique insights into the details of protein adsorption to porous media and will provide a valuable extension of the existing experimental methods for studies of mechanistic aspects of protein chromatography.

Acknowledgments

The authors are grateful to Barbara Jantsch and Markus Halfar for excellent assistance during the experimental work and to Dr Karol Lacki for making the micro-method for measuring adsorption isotherms available.

Notes

^*Please see color insert for Fig. 4.