(538d) Protein Elution within Polysaccharidic Stationary Phases for Ion-Exchange

Authors: 
Angelo, J. M., University of Delaware
Lenhoff, A. M., University of Delaware

Ion-exchange adsorbents modified with covalently attached or grafted

polymer layers have become more widely used in preparative chromatography. The

complex networks provided by polymeric carbohydrates such as cellulose and dextran

incorporate easily accessible microstructures with substantial binding capacities for

biomolecules. However, there exists an abnormal type of elution behavior in stationary

phases exhibiting these polymeric particle structures that may result in larger pool

volumes or product instability.

The anion and cation exchange moieties of commercially available cellulosic

and dextran-grafted agarose materials were characterized by their uptake and elution

profiles at differing total ionic strengths and pH conditions. Batch experiments were

performed alongside column level experiments to assess the transport and desorption

characteristics of several model proteins. Column elution profiles were analyzed to

determine the impact of loading factor with regard to elution pool volumes.

Additional mechanistic insight was sought using confocal laser scanning

microscopy techniques to gain a physical understanding of protein elution profiles

within single chromatographic particles under a variety of loading and elution

conditions. Abnormal profiles during elution within certain systems suggest alternative

mechanisms of protein expulsion and possibly increased protein-protein interactions

within the particle. Comparisons between polymer-modified and non-modified

materials were used to gauge the significance of pore structure in dictating functional

and mechanical characteristics of these stationary phases during elution.

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