Highlights► Phase behaviour of an aqueous two-phase system was visualised in counter-current chromatography. ► High density difference causes phase bilateral distribution anomalies. ► Low interfacial tension or settling time makes columns not retain stationary phase. ► Mobile phase flow orientation rule is modified.
Manufacturing high-value added biotech biopharmaceutical products (e.g. therapeutic proteins) requires quick-to-develop, GMP-compliant, easy-to-scale and cost effective preparatory chromatography technologies. In this work, we describe the construction and testing of a set of 5-mm inner diameter stainless steel toroidal columns for use on commercially available preparatory scale synchronous J-type counter-current chromatography (CCC) machinery. We used a 20.2 m long column with an aqueous two-phase system containing 14% (w/w) PEG1000 and 14% (w/w) potassium phosphate at pH 7, and tested a sample loading of 5% column volume and a mobile phase flow rate of 20 ml/min. We then satisfactorily demonstrated the potential for a weekly protein separation and preparation throughput of ca. 11 g based on a normal weekly routine for separating a pair of model proteins by making five stacked injections on a single portion of stationary phase with no stripping. Compared to our previous 1.6 mm bore PTFE toroidal column, the present columns enlarged the nominal column processing throughput by nearly 10. For an ideal model protein injection modality, we observed a scaling up factor of at least 21. The 2 scales of protein separation and purification steps were realized on the same commercial CCC device.
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