The binding of acidic proteins on Hydroxyapatite chromatography media is primarily based on the interaction of acidic groups of the protein with the Ca2+ ions on the resin matrix. Equilibrating the column with additional Ca2+ ions increases the amount of Ca2+ available for binding and allows higher binding capacities for acidic proteins. In this case study, the consequences of this secondary binding, of protein binding to Ca2+ ions loosely associated with the hydroxyapatite matrix is analyzed. The two distinctly different types of binding sites respond differently to wash and elution conditions and consequently can cause unpredictable or unstable operating behavior.  This presentation will highlight methods to identify binding to secondary Ca2+ ions as well as techniques to eliminate secondary binding to ensure simple column operation. Methodologies to stabilize a column operation in the presence of this secondary binding will also be discussed.

Secondary binding on hydroxyapatite chromatography media

239th National Meeting (2010)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.