Antibiotic purification from fermentation broths by counter-current chromatography: analysis of product purity and yield trade-offs

Abstract

Counter-current chromatography (CCC) is a low pressure, liquid-liquid chromatographic technique which has proven to be a powerful purification tool for the high-resolution fractionation of a variety of active pharmaceutical compounds. The successful integration of CCC into either existing or new manufacturing processes requires the predictable purification of target compounds from crude, fermentation-derived, feed streams. This work examines the feasibility of CCC for the purification of fermentation-derived erythromycin A (EA) from its structurally and chemically similar analogues. At the laboratory scale, the effect of feed pre-treatment using either clarified, forward extracted (butyl acetate) or back extracted broth on EA separation was investigated. This defined the degree of impurity removal required, i.e. back extracted broth, to ensure a reproducible elution profile of EA during CCC. Optimisation and scale-up of the separation studied the effects of mobile phase flow (2-40 ml.min(-1)) and solute loading (0.1-10 g) on the attainable EA purity and yield. The results in all cases demonstrated a high attainable EA purity (>97% w/w) with throughputs up to 0.33 kg.day(-1). Secondly, a predictive scale-up model was applied demonstrating, that from knowledge of the solute distribution ratio of EA (K(EA)) at the laboratory scale, the EA elution time at the pilot scale could be predicted to within 3-10%, depending upon the solute injection volume. In addition, this study has evaluated a "fractionation diagram" approach to visually determine the effects of key operational variables on separation performance. This resulted in accurate fraction cut-point determination for a required degree of product purity and yield. Overall, the results show CCC to be a predictable and scaleable separation technique capable of handling real feed streams.

Performance Characteristics of Countercurrent Separation in Analysis of Natural Products of Agricultural Significance

“…The relative proportions of the constituent solvents within a family can be modified almost endlessly; therefore, organized systems of solvent system family members have been developed in the CS literature – . Biphasic solvent systems composed of varying concentrations of hexane−ethyl acetate−methanol–water are used extensively to separate and isolate phytochemicals from extracts – . The HEMWat family of 17 hexane−ethyl acetate−methanol–water solvent systems has been constructed with a progression of polarity from most polar (+8) to least polar (−8), as shown in Table .…”

Countercurrent Separation of Natural Products

“…The sample was then partitioned on a 1 L type-J CS instrument. A maximum estimated product throughput of 410 g/day, with a yield and purity of 100% and 92%, respectively, was obtained. , Further studies with broth-derived antibiotics realized a throughput of up to 330 g/day with a greater than 97% purity . Flow rate and sample loading capacity are critical parameters in increasing throughput.…”

“…173,218 Further studies with broth-derived antibiotics realized a throughput of up to 330 g/day with a greater than 97% purity. 219 Flow rate and sample loading capacity are critical parameters in increasing throughput. At a certain, experimentally determined, point, increasing flow rate and/or loading capacity engenders a decrease in resolution.…”

Effect of Solution Polarity and Temperature on Adsorption Separation of Erythromycin A and C onto Macroporous Resin SP825

“…Booth (5,6) and our lab (7) suggested integrating counter-current chromatography (CCC) into erythromycin components separation processes. But the throughput of CCC is limited and the integration of CCC into downstream processes is too expensive.…”