Bio-Rad Sponsored Content

Mark Snyder1 and Esteban Freydell2 
1Bio-Rad Laboratories, Inc., Hercules, CA 94547; 2DSM Biotechnology Center — Downstream Processing, Delft, The Netherlands

High-Throughput Calcium Analysis of Hydroxyapatte Column Effluent 

Ceramic hydroxyapatite (CHT) is a mixed-mode chromatographic media widely used for the purification of proteins and monoclonal antibodies. CHT is a sintered form of hydroxyapatite (Ca5(PO4)30H), which is a form of calcium phosphate. The solubility of CHT increases at an acidic pH (pH <6.5) and it dissolves into its constituent ions (Ca2+, PO43-, OH-). Thus, the solubility of CHT can be estimated by measuring the concentration of the calcium ion, the phosphate ion, or the hydroxyl anion. Adding calcium and phosphate to buffers significantly enhances CHT lifetime under process conditions. The protocol described herein provides a rapid high-throughput method to quantify the total calcium concentration present in the effluent of a given CHT packed bed. The protocol has been modified such that it can be applied to either low or high buffering capacity samples, both typically encountered during purification protocols using CHT. 

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