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 (KEA) 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.