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 [1]. 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 [2]. 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: bersanetti@biofis.epm.br (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. [3], 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. [5] 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.
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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 [6]. 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 [5].
Mayer and Deckwer [4] 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 [2]. 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 [7], 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 [8].
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 [8]. 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.
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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. [5].
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.
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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.
The degradation rate constants of CA in aqueous solution
are lower than those obtained in fermentation medium, due
to the presence of other components of the medium, such as
ammonium compounds.
Expressions for the hydrolysis rate constant as a function of temperature (Arrhenius equation), constitute a significant result obtained in this work. Utilizing these equations it is possible to extrapolate other temperatures based on
conditions of choice in different production and purification
processes.
From the results obtained in this work, it is possible to
decide the best operating conditions of temperature and pH
under which clavulanic acid recovery process should be performed.
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