(315k) Ion Exchange Adsorption Process for in Situ Removal of the Unstable Β-Lactamase Inhibitor Clavulanic Acid from S. Clavuligerus Fermentation Broth

Clavulanic acid (CA) is one of the active ingredients in Augmentin - the world's best selling antibiotic medicine. It is a potent inhibitor of β-lactamases produced by some strains of pathogenic microorganism resistant to β-lactam antibiotics thereby enabling efficient treatment of infectious diseases. CA is produced in fermentative processes with Streptomyces clavuligerus growing on complex media. Maximal CA concentrations of 0.1 - 1 g/L using the wild type strain can be reached. The production process is hampered by the poor chemical stability of CA, decay takes place simultaneously with production. Furthermore experiments showed that CA biosynthesis is inhibited by clavulanic acid itself and also its decomposition products. We therefore aim for an improved production process by including in situ product removal (ISPR) and transferring CA in a stabilizing, e.g. chilled, environment. Promising methods under investigation are adsorption on ion exchange resins and Donnan dialysis. Several commercially available ion exchange resins show very good adsorption properties for clavulanic acid. However, stability of CA bound on an ion exchange resin is also low, therefore an ISPR process combining adsorption and desorption with chilled resin and elution phase would be necessary. A continuous process combining adsorption/desorption is the Donnan dialysis. In this process, an ion exchange membrane separates a diluted feed solution (e.g. 5 mM CA) and a strip solution containing a high concentration of so called driving ions (e.g. 1 M NaCl). Driving ions diffuse from the strip side to the feed side of the membrane thereby generating a driving force by which the feed ions are pumped to the stripping phase until the Donnan equilibrium is reached. First experiments showed that CA can be transported across ion exchange membranes. Pure CA solutions could be concentrated 11 fold in the strip phase. Using complex fermentation media supplemented with CA, partition coefficients in the range of 2 are reached due to competing ions in the media. Growth of Streptomyces clavuligerus on dialysed medium is not inhibited, showing that no essential medium components are withdrawn. The equilibrium and mass transfer characteristics of CA Donnan dialysis will be discussed in detail and a model enabling the design of an ISPR process will be developed. The effects of ISPR on the fermentative production of CA will be presented and discussed based on results obtained from our laboratory setup.