Hydroxyapatite is a naturally occurring mixed-mode chromatography media with unique selectivity. Its negatively charged phosphoryl groups (the P sites) interact with the amino residues on protein surface electrostatically, while the calcium ions in the crystal lattice (C sites) form chelation complexes with the acidic ligands on various biological molecules. Ceramic hydroxyapatite (CHT) is the hydrodynamically stable form of hydroxyapatite, which has been widely used for the manufacturing of protein therapeutics in the past three decades. The purification of acidic proteins with CHT chromatography has been challenging, as the charge characteristics of target proteins are very similar to impurities such as acidic proteins, nucleic acids, and endotoxins from the expression host. We have found that the concentrations of Ca2+ and inorganic phosphate in buffers have dramatic effects on the selectivity of CHT chromatography. By manipulating buffer composition and pH, we were able to modulate the interaction between the target protein molecule and CHT. An acidic recombinant enzyme, CDP-D-Glucose 4, 6-Dehydratase, was therefore purified from the E. coli extract in either flow-through or bind-and-elute mode. The acidic impurity clearance efficiencies of these two chromatography approaches are compared and presented herein.

Manipulating the selectivity of ceramic hydroxyapatite (CHT) for acidic protein purification

255th National Meeting (2018)

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