Firoz D. Antia

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.

Normal Phase High‐Performance Liquid Chromatography of Pneumocandins: In Situ Modification of Silica with l‐Proline To Separate Structural Analogues

Lipopeptides such as pneumocandin B0 are often produced by fermentation processes. Many compounds with similar structures (structural analogues), and hence similar physiochemical properties, are coproduced in the fermentation. We employed high performance liquid chromatography using silica gel as the stationary phase and a ternary ethyl acetate/MeOH/water mobile phase to separate pneumocandin B0 from these structural analogues. Despite extensive efforts to optimize this system, two key structural analogues, pneumocandin E0 and pneumocandin B5, continued to be poorly resolved from the main product peak (pneumocandin B0). As a result, feed load was restricted and productivity was limited. In situ modification of the silica gel stationary phase with l‐proline or other amino acids significantly enhances the resolution of the two key structural analogues from the compound of interest, enabling a two‐fold increase in productivity. Results of a systematic study showed that the amine group in l‐proline and other amino acids plays a key role in the modification of the surface of the silica gel to mediate the selectivity enhancement.