Biochemical Engineering Journal 23 (2005) 31–36 Kinetic studies on clavulanic acid degradation Patrı́cia A. Bersanettia,∗ , Renata M.R.G. Almeidab , Marlei Barbozac , Maria Lucia G.C. Araújoa , Carlos O. Hokkab a Department of Biochemistry and Technology, Institute of Chemistry, Universidade Estadual Paulista, Araraquara, SP, Brazil b Department of Chemical Engineering, Universidade Federal de São Carlos, São Carlos, SP, Brazil c Department of Chemical Engineering, Universidade de Ribeirão Preto, Ribeirão Preto, SP, Brazil Received 14 April 2004; received in revised form 9 September 2004; accepted 13 October 2004 Abstract Clavulanic acid (CA), a potent beta-lactamase inhibitor, is very sensitive to pH and temperature. It is produced by Streptomyces clavuligerus and to optimize both the fermentation step and the downstream process, the expression of the hydrolysis kinetics has to be determined. In the present work the CA degradation rate from various sources was investigated at temperatures of 10, 20, 25, 30 and 40 ◦ C and pH values of 6.2 and 7.0. The results showed that first-order kinetics explained very well the hydrolysis kinetics and the Arrhenius equation could be applied to establish a relationship between the degradation rate constant and temperature, at both pHs. It has been observed that CA from fermentation medium was much more unstable than that from standard solution and from a commercially available medicine. Also, it was observed that CA was more stable at pH 6.2 than at pH 7.0, irrespective of the CA source. © 2004 Elsevier B.V. All rights reserved. Keywords: Antibiotic; Clavulanic acid; Fermentation; Downstream processing; Degradation; Kinetic parameters 1. Introduction Many pathogenic bacteria secrete beta-lactamases, as a defense mechanism against beta-lactam antibiotics. Therefore, inhibitors of these enzymes are of potential clinical importance. Clavulanic acid (CA) is a potent beta-lactamase inhibitor and its application in conjunction with penicillin has proved to be a successful in countering the problem of resistance to antibiotics . This beta-lactam antibiotic, when used together with beta-lactamase sensitive penicillins, protects them against the hydrolysis of their beta-lactam ring by the enzymes and so renders them effective against betalactamase producing bacteria . It is known that clavulanic acid in its crude form is chemically unstable like other betalactam compounds. Several authors investigated the stability Abbreviation: CA, clavulanic acid Corresponding author. Present address: Department of Biophysics, Universidade Federal de São Paulo, Rua Tres de Maio 100, CEP 04044-020 São Paulo, SP, Brazil. Tel.: +55 11 5576 4455; fax: +55 11 5575 9617. E-mail address: [email protected] (P.A. Bersanetti). ∗ 1369-703X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2004.10.007 of CA in buffered aqueous solutions at various pHs [3–5]. These authors observed that CA degradation follows pseudofirst-order kinetics and that this degradation was highly influenced by catalysis caused by the buffering salts used to maintain constant pH. Haginaka et al. , who investigated CA stability at 35 ◦ C and an ionic strength (µ) of 0.5 at different pHs, observed that the degradation rate constant is highly dependent on the pH. The pH for CA maximal stability was estimated to be 6.39. The authors also studied the catalytic effects of buffer species at constant pH and ionic strength and concluded that the degradation rate constant increased with increasing buffer concentration. Bersanetti et al.  concluded that the stability of the antibiotic at 20 ◦ C and ionic strength (µ) 0.5 is highest at a pH around 6.0 and that degradation occurs more rapidly in basic solutions than in acidic solutions. The authors showed the relation between the hydrolysis rate constant of CA in aqueous solution and pH, in the range from 2 to 10 (Fig. 1). It was also found that ionic strength has no influence on this rate in the range studied. 32 P.A. Bersanetti et al. / Biochemical Engineering Journal 23 (2005) 31–36 tion broth (CA.3). Fermentation runs were carried out batchwise in a 5L Bioflo III Fermentor (New Brunswick Scientific Co. Inc., Edison, NJ, USA) at 28 ◦ C, pH 6.8 ± 0.1, 1 vvm and agitation speed ranging from 500 to 1000 rpm. The culture medium contained: glycerol (15.0 g); malt extract (10.0 g); Samprosoy 90 NB® (10.0 g); yeast extract (1.0 g) MgSO4 ·7H2 O (0.75 g); K2 HPO4 (0.80 g); soybean oil (1.0 g); trace element solution (1 mL) and distilled water (1 L), and was adjusted to pH 6.8 with NaOH/HCl . Imidazole and salts used to prepare the phosphate buffer were reagents of analytical grade. 2.2. Analytical methods Fig. 1. Effect of pH on the first-order rate constants for the clavulanic acid hydrolysis at 20 ◦ C and µ = 0.5 . Mayer and Deckwer  studied the simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivation on complex medium containing either soy meal extract or soy meal particles. The in vitro and in vivo CA stability were investigated. The authors concluded that the inactivation rate constants for the in vivo were considerably higher (2 to 10-fold) than those in vitro. While acid-catalyzed hydrolysis seemed to be responsible for most of the measured instability of CA in vitro, as indicated by the pH dependency of the degradation rate constant, one or several additional mechanisms are active in CA degradation during the stationary phase of cultures in the soy meal extract medium. In the work reported here, in order to determine the best conditions of temperature and pH to use in CA recovery processes, the stability of clavulanic acid at several temperatures (10–40 ◦ C) was investigated. Trials were carried out at two different pHs (6.2 and 7.0) which are in the range where the authors observed the lowest degradation rates [3,5]. The rate constants were determined, assuming that the process follows pseudo-first-order kinetics. Clavulanic acid from various sources was used, in aqueous solution or present in culture medium. It was possible, based on these results, to describe the dependence of the hydrolysis rate constant on temperature, utilizing the Arrhenius equation at each of the two pH values. 2. Materials and methods 2.1. Materials Clavulanic acid was obtained from three different sources: mixture of SiO2 /potassium clavulanate that was kindly provided by Gist Brocades, now DSM Anti-Infectives, Delft, The Netherlands (CA.1); potassium clavulanate from the pharmaceutical product Clavulin® (5 mL of oral suspension contain 62.5 mg of potassium clavulanate and 250 mg of amoxycillin), produced by SmithKline Beecham Laboratory, Rio de Janeiro, Brazil (CA.2); and clavulanic acid from fermenta- The clavulanic acid (CA.1 and CA.2) concentrations in aqueous solution were determined with an UV/Vis spectrophotometer (Pharmacia Biotech Ultrospec 2000) since there are no substances that could interfere with this method. The antibiotic reacted with an imidazole solution (60 g/L, pH 6.8) and the absorbance of the reaction product was measured at about 312 nm . The standard was the potassium clavulanate salt. Clavulanic acid concentrations of CA.3 were determined by high-performance liquid chromatography, as described by Foulstone and Reading , as the presence of other substances could interfere with UV analysis. The HPLC equipment with Photodiode Array detector (Waters 996 PDA) was operated with a reversed-phase column (C-18 µ-Bondapak 3.9 mm × 300 mm) maintained at 28 ◦ C, with a flow rate of 2.5 mL/min, and calibrated against solutions of the pharmaceutical product Clavulin® . The mobile phase was a 0.1 M KH2 PO4 buffer solution with 6% methanol, adjusted to pH 3.2 with phosphoric acid. 2.3. Experimental procedure The kinetic studies on clavulanic acid hydrolysis were carried out in 100 mL flasks at constant temperature, containing, in case of CA.1 or CA.2, 20 mL of a clavulanic acid solution to give an initial concentration of 40 mg/L, 30 mL of 0.04 M phosphate buffer and potassium chloride to adjust the ionic strength to 0.5. In the experiments with CA.3, 40 mL of filtered fermented medium and 10 mL of 0.04 M phosphate buffer were used. Samples of 0.5 mL were withdrawn periodically and the concentration of undegraded clavulanic acid was determined. The temperatures and pH values used are shown in Table 1. The pH range studied was based on literature data [3,5] showing that clavulanic acid has a higher stability between Table 1 Temperatures and pH values of the reaction flasks CA.1 and CA.2 CA.3 CA.1, CA.2 and CA.3 pH Temperature (◦ C) 6.2 6.2 7.0 10, 20, 25 and 30 10, 20, 30 and 40 10, 20, 30 and 40 P.A. Bersanetti et al. / Biochemical Engineering Journal 23 (2005) 31–36 pHs 6 and 7. The temperatures chosen were suitable for industrial clavulanic acid recovery processes. The ionic strength of 0.5 was based on earlier work of a similar nature on other beta-lactam compounds . 3. Results and discussion 3.1. Degradation of clavulanic acid in aqueous solution The experimental results in Figs. 2 and 3, for pHs 6.2 and 7.0, respectively, show that clavulanic acid degradation, in aqueous solution, follows a pseudo-first-order kinetics (Eq. (1)), as other beta-lactam compounds . The straight lines in these figures are the linear fit for each temperature − dC = kC dt (1) The degradation rate constants (k), estimated by linear regression of the experimental data in Figs. 2 and 3, are given in Tables 2 and 3 for pHs 6.2 and 7.0, respectively, for each 33 temperature. It can be observed that the values of the rate constant clearly increases with temperature, however, for each temperature, the k values for CA.1 and CA.2 are very similar. Comparing the rate constants of CA.1 at pH 6.2 and various temperatures, in Table 2 with those at pH 7.0, in Table 3, it is observed that the corresponding rate constants at pH 7.0 are considerably higher than those obtained at pH 6.2. This difference increases as the temperature rises (1.3-fold at 10 ◦ C to 2.3-fold at 30 ◦ C). The same occurs with CA.2, as can be seen by comparing Table 2 with Table 3 (1.7-fold at 10 ◦ C to 2.3-fold at 30 ◦ C). Thus, as observed by other authors, clavulanic acid has a higher stability at a pH around 6.0; in addition the stability decreases as temperature increases, irrespective of the source of the antibiotic. Still analyzing the values of k in Tables 2 and 3, it is observed that, at temperatures above 10 ◦ C, the stability of CA.2 is slightly higher than CA.1 at both values of pH, probably due to some stabilizing compounds present in the pharmaceutical product formula. However, this difference is not remarkable enough to impair the use of a same value of k for both cases. Fig. 2. Clavulanic acid degradation, pH 6.2, at various temperatures: (a) CA.1; (b) CA.2. Fig. 3. Clavulanic acid degradation, pH 7.0, at various temperatures: (a) CA.1; (b) CA.2. 34 P.A. Bersanetti et al. / Biochemical Engineering Journal 23 (2005) 31–36 Table 4 Rate constant and the half-life obtained from the degradation of CA.3 at pH 6.2 Table 2 Degradation rate constant at pH 6.2 of CA.1 and CA.2 Temperature (◦ C) (correlation coefficient) 10 (r2 = 0.958) 20 (r2 = 0.997) 25 (r2 = 0.990) 30 (r2 = 0.998) a b k (h−1 ) Half-life (h) CA.2 Temperature (◦ C) (correlation coefficient) k (h−1 ) CA.1 0.00177 ± 0.00020 0.00383 ± 0.00011a 0.00535 ± 0.00017 0.00723 ± 0.00012 0.00196 ± 0.00023 0.00336b 0.00409 ± 0.00019 0.00614 ± 0.00042 10 (r2 = 0.993) 20 (r2 = 0.969) 30 (r2 = 0.993) 40 (r2 = 0.984) 0.01324 ± 0.00048 0.02186 ± 0.00157 0.02940 ± 0.00087 0.03838 ± 0.00443 52.3 31.7 23.6 18.1 Result obtained from Bersanetti et al. . This value was estimated by Arrhenius equation (see Table 6). 3.2. Degradation of clavulanic acid from fermentation Table 3 Degradation rate constant at pH 7.0 of CA.1 and CA.2 Temperature (◦ C) (correlation coefficient) 10 (r2 = 0.983) 20 (r2 = 0.983) 30 (r2 = 0.996) 40 (r2 = 0.999) k (h−1 ) CA.1 CA.2 0.00234 ± 0.00018 0.00668 ± 0.00051 0.01672 ± 0.00064 0.03907 ± 0.00071 0.00344 ± 0.00032 0.00654 ± 0.00026 0.01422 ± 0.00039 0.03447 ± 0.00049 In order to avoid contamination of the fermentation medium of CA.3, which could interfere in the antibiotic stability results, in this case the degradation process was studied for only 10 h, approximately. The linear fits of the experimental data, utilized to determine the hydrolysis rate constants at pH 6.2, are shown in Fig. 4(a) for 10 and 20 ◦ C and (b) for 30 and 40 ◦ C, respectively. Table 4 shows the values of k and the half-life obtained from the degradation of CA.3 at pH 6.2. The straight lines fitted to the experimental data and used to calculate the hydrolysis rate constants at pH 7.0 are plotted Fig. 4. Clavulanic acid (CA.3) degradation at various temperatures, pH 6.2: (a) 10 and 20 ◦ C; (b) 30 and 40 ◦ C. Fig. 5. Clavulanic acid (CA.3) degradation at various temperatures, pH 7.0: (a) 10 and 20 ◦ C; (b) 30 and 40 ◦ C. P.A. Bersanetti et al. / Biochemical Engineering Journal 23 (2005) 31–36 Table 5 Rate constant and the half-life obtained from the degradation of CA.3 at pH 7 Temperature (◦ C) (correlation coefficient) k (h−1 ) Half-life (h) 10 (r2 = 0.987) 20 (r2 = 0.992) 30 (r2 = 0.964) 40 (r2 = 0.971) 0.01443 ± 0.00087 0.02397 ± 0.00086 0.04152 ± 0.00290 0.05490 ± 0.00346 48.0 28.9 16.7 12.6 Table 6 Arrhenius equations for each source of CA at pHs 6.2 and 7.0 CA.1 + CA.2 CA.3 pH 6.2 pH 7.0 k = 5.74 × 105 e−46,048/RT k = 3.91 × 102 e−23,979/RT k = 3.83 × 109 e−66,025/RT k = 8.31 × 103 e−30,969/RT in Fig. 5(a) and (b), for 10–20 and 30–40 ◦ C, respectively. Table 5 shows the values of k and the half-life obtained from the degradation of CA.3 at pH 7.0. It was observed, in Tables 4 and 5, that the degradation rate constant again increases with temperature increasing at both pH values and the stability of CA.3 is higher at pH 6.2 than at pH 7.0, as expected. Comparing Table 2 with Table 3 and Table 4 with Table 5, note that the degradation rate constants of CA.3 experiments are higher than those obtained with clavulanic acid in aqueous solution at both pHs 6.2 and 7.0. This effect is probably due to the presence of many components, such as ammonium compounds, in fermentation medium that increase CA instability. The difference between the hydrolysis rate constants of CA in aqueous solution and in fermentation medium is higher at 10 and 20 ◦ C than at higher temperatures, 30 and 40 ◦ C, at both pHs. 3.3. Dependence of rate constant on temperature From the values obtained for the hydrolysis rate constants, it was possible to establish a relationship between the value of 35 k and temperature by fitting the data to the Arrhenius equation (Eq. (2)), which relates rate constants to temperature. Since the k values for CA.1 and CA.2 samples were similar, the degradation constant for both cases were analyzed together. The resulting expressions, for each source of CA at each pH are displayed in Table 6. k = A e−Ea /RT (2) where k is the reaction constant (h−1 ), A the frequency factor (h−1 ), Ea the activation energy (J/mol), R the gas constant (8.314 J/mol K), and T the temperature (K). These results displayed in Table 6 can be used to determine the degradation rate constant at other temperatures and establish the optimum temperature conditions of the production and purification processes. As shown in Table 6, CA from fermentation broth, CA.3, presents lower activation energy of the degradation reaction, probably due to the presence of other substances in the medium, as also shown by Roubos et al. In fact, these substances increase the number of collisions between CA molecules and the impurities molecules. The increase of activation energy due to pH increase must be due to lower acid catalytic activity on the degradation reaction. Utilizing the Arrhenius equations it is possible to determine at which temperatures (TE ) the CA degradation rate constants are equal at each of the two pHs. These temperatures are at the crossing points in Fig. 6(a) and (b) for CA.1 + CA.2 and CA.3, respectively, and are observed to be −1 and 5 ◦ C for CA.1 + CA.2 and CA.3, respectively. At temperatures below these values, the CA stability is higher at pH 7.0 than at pH 6.2. However, to work in this range of temperature of CA.1 + CA.2, it would be necessary to use frozen solutions, which are unsuited to the conventional production and separation processes. In the case of CA.3, it is possible to work with separation/purification process at 5 ◦ C at pH Fig. 6. Arrhenius plots showing temperatures at which k values are equal at both pHs: (a) CA.1 + CA.2, TE = −1 ◦ C; (b) CA.3, TE = 5 ◦ C. 36 P.A. Bersanetti et al. / Biochemical Engineering Journal 23 (2005) 31–36 7.0, but further studies of refrigeration costs are necessary to evaluate the actual advantages in working at this temperature. Acknowledgement The authors gratefully acknowledge the financial support of the Brazilian Research Funding Institution FAPESP (Process 99/07693-2, 98/11596-0 and 99/03279-7). 4. Conclusions References Clavulanic acid has a higher stability at pH 6.2 than at pH 7.0, both in aqueous solution and in vitro, irrespective of the source of CA. In the case of CA.1 and CA.2 this difference is more marked at temperatures above 10 ◦ C. At temperatures below −1 and 5 ◦ C for CA.1 + CA.2 and CA.3, respectively, this effect of the pH on the stability of CA is reversed, according to the Arrhenius equations fitted to the data. 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