The resolution of two clinical agents, bleomycin and desferrioxamine B, from a Streptomyces verticillus fermentation mixture using multi-dimensional immobilised metal ion affinity chromatography

Abstract

The multi-step process of extracting and purifying secondary metabolites as pharmaceutics from bacterial culture often represents the highest overall cost in production, and can require large volumes of organic solvents. In this work, we describe an aqueous-compatible, streamlined and selective method for the simultaneous isolation of two clinical agents, bleomycin (BLM) and desferrioxamine B (DFOB), from a standard solution, and from native Streptomyces verticillus culture. The method used two in-series columns containing immobilised metal ion affinity chromatography (IMAC) resin charged with Yb(III) (upper column) or Cu(II) (lower column), designed to exploit differences between the coordination chemistry of DFOB and BLM. Application of this multi-dimensional (MD)-IMAC configuration to a standard solution containing DFOB and BLM resulted in a complete resolution, with the high-yielding (∼90%) isolation of DFOB on the Yb(III)-charged resin, and BLM on the Cu(II)-charged resin. The resolution was dependent upon the metal ion, the denticity of the immobilised chelate and the column order. The method was successful in the complete resolution of native DFOB and BLM from the complex S. verticillus fermentation mixture. DFOB eluted from the Yb(III) resin as the free ligand. BLM eluted from the Cu(II) resin as BLM A₂, Cu(II)–BLM A₂ and Cu(II)–BLM B₂. The optimised Yb(III)–IMAC format had a DFOB binding capacity (≥95%) of 8 μmol mL⁻¹, which is significantly greater than previously described formats. This work has demonstrated the potential of MD-IMAC for improved pharmaceutics processing of agents from fermentation through process intensification, and for reducing the environmental, occupational and financial costs of the use of organic solvents.