Process-scale reversed-phase high-performance liquid chromatography purification of LL-E19020α, a growth promoting antibiotic produced by Streptomyces lydicus ssp. tanzanius

Abstract

LL-E19020α is a novel antibiotic produced by fermentation of the soil microorganism Streptomyces lydicus spp. tanzanius. The compound is highly effective in inducing increasing in weight gain and feed conversion efficiency in livestock. In order to obtain kilogram quantities of the material for field trials, pilot plant scale fermentations (up to 7500 1) were carried out. The antibiotic was recovered from the fermentation broth by solvent extraction. The resultant crude extract was subjected to reversed-phase (C 18) chromatography on a process-scale high-performance liquid chromatography (HPLC) unit. The heart of the instrumentation is the Millipore Kiloprep® chromatograph with the standard 12-1 cartridge column. The laboratory housing the chromatograph has been specifically designed for this work. Tanks for mobile phase preparation are mounted on load cells for precise measurement of components. In this explosion-proof laboratory, all solvent handling areas are well ventilated and a separate breathing air system is provided for the operators. For the purification of the LL-E19020 antibiotics, the mobile phase consisted of a gradient of acetonitrile in 0.1 M ammonium acetate at pH 4.5. The effluent was monitored by UV absorbance at 325 nm. Fractions were collected across the peaks of interest and these were analyzed by analytical HPLC. The maximum yield of LL-E19020α obtained in a single run was approximately 100 g. The antibiotic was recovered from the mobile phase by extraction with methylene chloride. The methylene chloride phase was concentrated under reduced pressure to yield a gummy residue which was finally freeze-dried from tertiary butanol to yield an off-white solid suitable for blending with various feed components.