Use of an alternative scale-down approach to predict and extend hydroxyapatite column lifetimes

Ceramic hydroxyapatite (CHT) chromatography offers unique selectivity for protein purification. However, columns composed of CHT, a crystalline form of calcium phosphate, often suffer from short column lifetimes, particularly under acidic operating conditions. In this paper, CHT was used under slightly acidic conditions (pH 6) for the production scale purification of a recombinant protein. Under these conditions, the packing quality of production scale CHT columns (45 cm diameter) degraded after 5-10 cycles of operation. This was not reproduced using a conventional scale-down chromatography model, in which a constant column bed height was maintained across scales. Thus, an alternative scale-down model was developed to better predict the lifetime of large scale CHT columns. The alternative approach, which utilized a constant column diameter-to-height aspect ratio, was able to predict column failure that approximated that of the manufacturing scale column. The alternative scale-down approach was then used to test alternate buffer formulations that significantly improved the CHT column lifetime. Screening studies, which assessed the effects of mobile phase pH and composition on the dissolution (weight loss) of CHT, were used to identify the alternative mobile phase formulations. Results from the study showed that slight changes to the existing mobile phase compositions significantly increased the column lifetime, from approximately 10 cycles to approximately 65 cycles of use, without altering the purification of the recombinant protein. The alternative scale-down model, together with relatively rapid mobile phase screening studies, provides a practical approach for predicting and optimizing the useful lifetime of CHT columns for large scale applications.