Stability of clavulanic acid in PEG/citrate and liquid–liquid extraction in aqueous two-phase system

Abstract

␤-Lactamases are enzymes responsible for the hydrolysis of ␤-lactam antibiotics, being produced by several pathogenic bacteria. Clavulanic acid is a commercially and clinically important ␤-lactamase inhibitor, its extraction being possible by the application of aqueous two-phase system. In this study, clavulanic acid stability was investigated at different molar mass PEG (400, 1 000 and 20 000 g mol −1) and at different citrate concentrations (5 and 20%) PEG/citrate aqueous-two phase systems (ATPS), under different pH values (4.0-8.0). Clavulanic acid extraction was also evaluated. Low citrate concentration and PEG 20 000 (g mol −1) ATPS, at pH 6.0, were shown to be the systems that presented the highest clavulanic acid stability. Based on this, a factorial design 2 2 was used to evaluate CA extraction, being PEG (20, 25 and 30%) and citrate (5, 10 and 15%) concentrations the parameters evaluated. Clavulanic acid was extracted into the PEG-rich phase (133.66 mg L −1), the highest values of the partition coefficient and yield being K = 5.92, Y = 103.53%, respectively The ATPS was not only effective on clavulanic acid extraction, but also its degradation was minimal. These results clearly indicate that ATPS can be successfully applied as a first step for the purification of clavulanic acid.

Purification of clavulanic acid produced by Streptomyces clavuligerus via submerged fermentation using polyethylene glycol/cholinium chloride aqueous two-phase systems

“…Considering that [Ch]Cl exhibits a lower ionic strength than the most common inorganic salts previously studied, the negligible influence of [Ch]Cl ionic strength on the CA degradation observed in this work is easily understood. In addition, this negligible effect agrees with previous findings of Laidler [51], who highlighted that the salts' ionic strength has no relevant influence on the rate of hydrolysis reactions [51], and by Carneiro-da-Cunha et al, in which the citrate concentration effect over CA degradation was negligible [50]. These results support the high biocompatibility of [Ch]Cl, allowing the most appropriate adjustment of the [Ch]Cl composition in each ATPS to reach the best extraction performance without compromising the CA structural integrity.…”

“…It is important to notice that the polymer type (PEG-600-or PEG-550-OMe) used does not affect significantly the extraction of CA, being the system based in 60 wt% of polymer and 30 wt% of [Ch]Cl the only exception. Meanwhile, the partition coefficient data obtained in this study are lower than those previously obtained for the use of traditional PEG/inorganic salts-based ATPS [7,45,50,53].…”

“…In addition, CA is quite stable in presence of polymeric aqueous solutions, as already demonstrated in literature for several concentrations of aqueous solutions of PEG with variable molar mass (from 2000 until 20000 g mol À1 ) [6,50] and NaPA [6]. Taking into account the CA stable character in presence of polymeric aqueous solutions, and the low CA degradability in presence of [Ch]Cl, PEG-600/[Ch]Cl-PEG-550-OMe/[Ch]Cl-and PEG-600/NaPA-8000/[Ch] Cl-based ATPS were further investigated towards the partitioning and recovery of CA produced via fermentation.…”

“…CA is more stable in neutral or slightly acidic solutions (pH between 6 and 7.2), and temperatures close to 25 C or lower [49]. However, the aqueous solutions with high salt concentration can induce a prejudicial effect on the drug stability, causing its degradability [50]. It was in this context that some experiments were carried out to investigate the effect of different [Ch]Cl aqueous solutions towards the CA stability, being the main results depicted in Fig.…”

Dextran sulfate/Triton X two-phase micellar systems as an alternative first purification step for clavulanic acid

“…One of the most important resistance mechanisms exhibited by a variety of bacteria is their ability to produce b-lactamases, enzymes that inactivate the b-lactam ring of penicillins and cephalosporins antibiotics [2,3]. To overcome this resistance, the use of clavulanic acid (CA) in the form of potassium salt combined with amoxicillin (Augmentin TM ) or ticarcillin (Timentin TM ) has been employed.…”