Bleomycin A2and B2Purification by Flash Chromatography for Chemical and Biochemical Studies

Abstract

The clinically used formulation of the anticancer antibiotic, Blenoxane, is a mixture of bleomycin congeners. A new approach to separating the major A2 and B2 congeners has been developed utilizing the flash chromatography technique. A 5-6-inch column of fine mesh silica gel with a solvent system of 1% ammonium formate:methanol (2:3) was used. Low air pressure was applied to the column to increase the flow rate such that separation was complete in approximately 20 min. Reverse phase size exclusion gravity chromatography with Sephadex G-15 column bedding was an effective, rapid procedure for removal of the 1% ammonium formate, the lowest percentage practical for separating the bleomycins. This separation approach does not damage the antibiotics, as demonstrated by NMR spectroscopy, thin layer chromatography, and DNA cleaving activity. Although not as useful for detection of trace amounts of the drug in biological systems as some of the known HPLC methods, this method is excellent for separating large quantities of the drug (8-32 mg) in order to obtain congeners pure enough for synthetic, biochemical, and biophysical studies.