384564 Protein Elution within Polysaccharidic Stationary Phases for Ion-Exchange
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.