Optimization of the fractionation and recovery of polyketide antibiotics by countercurrent chromatography

The polyketide antibiotics are a large and diverse range of natural products exhibiting a wide range of antimicrobial activities. The biosynthesis of these compounds results in the formation of analogues of the main antibiotic that are structurally and chemically very similar. These pose a significant separation problem, particularly on a large scale. In this work, we have investigated the fractionation and recovery of erythromycin A (EA) from its analogues by countercurrent chromatography (CCC). In particular, we studied the effect of increasing mobile phase flow rate (2-10 mL.min(-1)) and solute loading (0.1-1.0 g) on various chromatographic parameters, such as stationary phase retention, solute partition coefficients, and column efficiency, together with the estimated throughput of the process. Experiments were performed on a Quattro J-type coil planet centrifuge (94.3 or 101.1 mL PTFE coil, 800 rpm) using a quaternary phase system, comprising hexane/ethyl acetate/methanol/water (1.2/2.0/2.0/1.0 v/v). Under optimal conditions, at a mobile phase flow rate of 8 mL.min(-1) and an injected solute mass of 0.6 g, EA could be obtained with a purity and yield of 97% w/w and 100% w/w, respectively. The maximum solute throughput in this case was estimated to be 0.96 Kg.(L-coil.day)(-1), which represented a 17 fold increase over the starting conditions identified during earlier method development studies. The results provide an encouraging basis for the subsequent application of CCC technology to the separation of novel recombinant polyketides, currently being developed by combinatorial biosynthesis techniques.