Marlei Barboza

Influence of temperature on the kinetics of adsorption and desorption of clavulanic acid by ionic exchange

Abstract Beta-lactamases are enzymes that deactivate penicillins and cephalosporins by hydrolyzing their beta-lactam rings, producing penicilloic acid derivatives and, in the case of cephalosporins, analogous degradation products. Clavulanic acid (CA) is a beta-lactam antibiotic produced by Streptomyces clavuligerus , which has a potent beta-lactamase inhibiting activity. This antibiotic displays only weak antibacterial activity, therefore rendering it unsuitable for use by itself. However, its use in combination with certain penicillins (amoxycillin and ticarcillin) is effective in clinical use. The recovery process of CA involves steps such as adsorption, ion exchange chromatography or liquid–liquid extraction, but with low yields. Thus, in the case of CA, an investigation of the influence of temperature on the adsorption process is very important because of the unstable structure of this antibiotic, which degrades rapidly under normal processing conditions. In the work reported on herein, the influence of temperature on the CA adsorption process was investigated, involving equilibrium and kinetic studies and carrying out batch shake experiments with the ion exchanger resin Amberlite IRA 400 at four different temperatures. A model of the CA adsorption process, taking into account mass transfer limitation, is proposed. An estimate of the thermodynamics of the adsorption process was made based on van’t Hoff’s equation.

Kinetic studies of clavulanic acid recovery by ion exchange chromatography

Clavulanic acid (CA) is a beta-lactamase inhibitor produced by strains of Streptomyces clavuligerus. Nowadays, the combination of CA with amoxycillin is the most successful example of the use of a beta-lactam antibiotic sensitive to beta-lactamases together with an inhibitor of these enzymes. Clavulanic acid is purified from fermentation broth by a series of steps consisting mainly of two-phase separation processes such as liquid–liquid extraction, adsorption or ion-exchange chromatography, among others. Amberlite IRA 400, a strong anion-exchange resin, has a very high adsorption capacity for CA (Mayer et al. 1997). This resin can be pre-treated with NaCl (chloride cycle), to remove selectively only those anions, which are able to displace chloride from the resin or with NaOH (hydroxyl cycle), to remove all species of anions. In order to decide the best operating conditions for CA recovery by ion-exchange resins and then to construct a model of this separation process, batch experiments were conducted using Amberlite IRA 400 in the chloride cycle. These runs were carried out in a 200 ml stirred tank, at two different initial solution pH, 6.2 and 4.0; the temperature was maintained at 10 °C and 20 °C during adsorption and 30 °C during the desorption step. It was possible, on the basis of these batch results, to model the separation process, including the adsorption kinetics, equilibrium data and mass transfer limitations.

Separation of clavulanic acid from fermented broth of amino acids by an aqueous two-phase system and ion exchange adsorption

The clavulanic acid is a substance which inhibits the β-lactamases used with penicillins for therapeutic treatment. After the fermentation, by-products of low molecular weight such as amino acids lysine, histidine, proline and tyrosine are present in the fermented broth. To remove these impurities the techniques of extraction by an aqueous two-phase system of 17% polyethylene glycol molecular weight 600 and 15% potassium phosphate were used for a partial purification. A subsequent ion-exchange adsorption was used for the recuperation of the clavulanic acid of the top phase and purification getting a concentration factor of 2 and purification of 100% in relation to the amino acids lysine, histidine, proline and tyrosine.

Power Input and Oxygen Transfer in Fed-Batch Penicillin Production Process

Fed-batch experiments in a 20 l fermentor utilizing Penicillium chrysogenum IFO 8644 were conducted using a medium containing sucrose and corn steep liquor as main components. The runs were interrupted at different stages of the process – 24, 48, 72, 96 and 120 h — and broths were transferred to an agitated vessel. Power consumption under gassed and ungassed broths and volumetric oxygen transfer coefficient, kLa, were measured for various agitation speeds and aeration rates. The rheological characteristics of each broth were also determined. Power number — modified Reynolds number plots were similar to those for other non -Newtonian fluids. Michel and Miller correlation was applied for predicting gassed power drawn for all broths. A general correlation for kLa, as a function of operating variables and apparent viscosity for broths of different cultivation ages was obtained.

Preliminary Studies for Cephamycin C Purification Technique

A study was made for purification of cephamycin C from fermentation of Streptomyces clavuligerus. Initially, the culture broth was clarified by microfiltration and ultrafiltration, after which the resulting permeates were subjected to nonspecific adsorption and ion-exchange chromatography on resin columns. The antibiotic activity was measured by the biological method at each stage by assaying its activity against the Escherichia coli ESS, super sensitive to β-lactam antibiotic. The purification processes were assessed in relation to the variables affecting each step. The purification efficiency by nonspecific adsorption was monitored by UV spectrophotometry, while the ion-exchange adsorption fractions were assessed by NMR spectroscopy. Some of the fractions obtained during purification were also analyzed by mass spectrometry (LC/MS and LC/MS/MS) to identify the cephamycin C molecule. These preliminary results proved the process feasibility.

Continuous Clavulanic Acid Adsorption Process

Adsorption kinetics and equilibrium data of clavulanic acid, a beta-lactam antibiotic, on ion-exchange resin Amberlite IRA 400 were utilized to carry out the modeling and simulation of a continuous adsorption process. These simulations allowed the estimation of yield, concentration, and purification factors of the process utilizing the product final concentration. Experimental runs of this process were carried out using the conditions pointed out by simulation studies. Comparison of the experimental results and those calculated by the proposed model showed that the model could describe very well the main features of the continuous process.

General relationship for volumetric oxygen transfer coefficient (kLa) prediction in tower bioreactors utilizing immobilized cells

Abstract A general relationship for prediction of the volumetric oxygen transfer coefficient (kLa) in a tower bioreactor utilizing immobilized Penicillium chrysogenum as function of air superficial velocity, suspension rheological parameters and liquid physical properties is proposed in this study. The relationship was applied to three different systems and a good agreement between the calculated values and the experimental data was obtained.

An Alternative Methodology for Determination of Cephamycin C from Fermentation Broth

Cephamycin C is a β-lactam antibiotic used as a raw material in several commercial antibiotics. The production and purification process of this antibiotic requires a fast, simple and accurate method to quantify it. In this paper, it was developed a high-performance liquid chromatography method to determine cephamycin C concentration, in order to offer an option for the traditional, but laborious, bioassay method normally employed. A method to obtain a calibration curve using the bioassay and cephalosporin C as standard was also proposed. The method showed more efficiency in determining cephamycin C than the bioassay, and it was simpler and faster to execute.

Ácido clavulânico e cefamicina c: uma perspectiva da biossíntese, processos de isolamento e mecanismo de ação

The present article reviews different aspects of the chemistry of two widely used β-lactam antibiotics Clavulanic Acid and Cephamycin C. The article discusses important details of the biosynthesis of these compounds, their action mechanism and, principally, the methods employed in their isolation and purification, in accordance with the available literature. Despite the large quantity of available articles and patents concerning β-lactam antibiotics, those which describe the isolation and purification of Clavulanic Acid and Cephamycin C are rare. Overall, the intention of this article is to discuss the up-to-date scientific research related to the compounds under review.

Optimization of the precipitation of clavulanic acid from fermented broth using t-octylamine as intermediate

Abstract - This work describes the use of clavulanic acid (CA) precipitation as the final step in the process of purification of CA from fermentation broth as an alternative to conventional methods employed traditionally. The purpose of this study was to use a stable intermediate (t-octylamine) between the conversion of CA to its salt form (potassium clavulanate), thereby enabling the resulting intermediate (amine salt of clavulanic acid) to improve the purification process and maintain the stability of the resulting potassium clavulanate. To this end, response surface methodology was employed to optimize the precipitation step. For the first reaction, five temperatures (6.6 to 23.4 °C), concentrations of clavulanic acid in organic solvent (6.6 to 23.4 mg/mL) and t-octylamine inflow rates (0.33 to 1.17 drop/min) were selected based on a central composite rotatable design (CCRD). For the second reaction, five temperatures (11.6 to 28.4 °C), concentrations of clavulanic acid amine salt in organic solvent (8.2 to 41.8 mg/mL) and concentrations of potassium 2-ethylhexanoate (0.2 to 1.2 molar) were also selected using CCRD. From these results, precipitation conditions were selected and applied to the purification of CA from the fermentation broth, obtaining a yield of 72.37%.