Preparative high-performance liquid chromatography of echinocandins

Abstract

The isolation of fermentation derived small bioactive molecules remains extremely challenging due to the presence of many analogues with similar physicochemical behavior. Removal of analogue impurities typically involves crystallization and/or preparative HPLC. The normal-phase preparative HPLC for the purification of fermentation derived echinocandins is described. Resolution of key impurities from the product of interest, pneumocandin B o, is accomplished using a ternary ethyl acetate–methanol–water mobile phase with silica gel as the sorbent. Plate counts, measured with small molecules, show that the column efficiency is excellent under the operating conditions despite the use of an irregular silica and unusually high levels (greater than 6%) of water in the mobile phase. The results of optimization studies indicate the product solubility, retention and resolution of key analogue impurities are strong functions of the ternary mobile phase composition. The normal-phase HPLC process was optimized by carrying out eluent flow-rate (linear velocity) and column loading studies. The results of these experimental studies indicate that both yield and productivity are a function of linear velocity and product loading and that a tradeoff exists between these two parameters.