Freeze-dried snake antivenoms formulated with sorbitol,
sucrose or mannitol: Comparison of their stability in an
accelerated test
María Herrera
a,*, Virgilio Tattini Jr.
b, Ronaldo N.M. Pitombo
b,
Jos
e María Guti
errez
a, Camila Borgognoni
b, Jos
e Vega-Baudrit
c,
Federico Solera
c, Maykel Cerdas
a,
Alvaro Segura
a, Mauren Villalta
a,
Mari
angela Vargas
a, Guillermo Le
on
aaInstituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jose, Costa Rica
bDepartment of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of Sao Paulo, Av. Prof. Lineu
Prestes, 580, Bloco 16, CEP05508-900, Sao Paulo, SP, Brazil
cLaboratorio Nacional de Nanotecnología, LANOTEC-CENAT, San Jose, Costa Rica
a r t i c l e i n f o
Article history:
Received 31 March 2014
Received in revised form 22 July 2014 Accepted 24 July 2014
Available online 1 August 2014
Keywords:
Antivenom Freeze-drying Sorbitol Mannitol Sucrose Stability
a b s t r a c t
Freeze-drying is used to improve the long term stability of pharmaceutical proteins. Sugars and polyols have been successfully used in the stabilization of proteins. However, their use in the development of freeze-dried antivenoms has not been documented. In this work, whole IgG snake antivenom, purified from equine plasma, was formulated with different concentrations of sorbitol, sucrose or mannitol. The glass transition temperatures of frozen formulations, determined by Differential Scanning Calorimetry (DSC), ranged between 13.5C and41C. In order to evaluate the effectiveness of the different stabilizers, the freeze-dried samples were subjected to an accelerated stability test at 40 ±2 C and 75±5% relative humidity. After six months of storage at 40C, all the formulations pre-sented the same residual humidity, but significant differences were observed in turbidity, reconstitution time and electrophoretic pattern. Moreover, all formulations, except anti-venoms freeze-dried with mannitol, exhibited the same potency for the neutralization of lethal effect ofBothrops aspervenom. The 5% (w:v) sucrose formulation exhibited the best stability among the samples tested, while mannitol and sorbitol formulations turned brown. These results suggest that sucrose is a better stabilizer than mannitol and sorbitol in the formulation of freeze-dried antivenoms under the studied conditions.
©2014 Elsevier Ltd. All rights reserved.
1. Introduction
Freeze-drying or lyophilization is the most commonly used method for preparing solid proteins which are phys-ically or chemphys-ically unstable in aqueous solution. However,
most proteins are sensitive to lyophilization due to the stress of freezing and drying that can cause irreversible damage to the protein structure and biological activity (Heller et al., 1999; Sarciaux et al., 1999). The effectiveness of this technology in the stabilization of biopharmaceutical products is the result of the combination of optimizing the formulation and controlling the process. Formulation optimization is focused on the use of disaccharides as sta-bilizers, together with bulking agents, such as mannitol and glycine (Imamura et al., 2003; Sharma and Kalonia, 2004).
*Corresponding author. Tel.:þ506 2511 7878; fax:þ506 2292 0485.
E-mail addresses: [email protected], mariaherrv@hotmail. com(M. Herrera).
Contents lists available atScienceDirect
Toxicon
j o u r n a l h o m e p a g e : w w w .e l se v i e r. co m/ lo ca t e / t o x i co n
http://dx.doi.org/10.1016/j.toxicon.2014.07.015
On the other hand, the optimization of the process involves controlling the freezing and drying stages for each formu-lation developed.
Immunoglobulins are a group of proteins relevant as pharmaceuticals and as diagnostic agents. These proteins are prone to forming aggregates and undergo other phys-ical and chemphys-ical modifications during manufacturing and long term storage, which may cause loss of activity (Maury et al., 2005). The immunoglobulins G (IgGs) constitute the active principle of antivenoms. Snake antivenoms are considered the only scientifically proven therapy against snakebite envenomation (Bon, 1996), and they are pro-duced from the plasma of animals immunized with a venom or a mixture of venoms.
Snakebite envenomation is an important neglected tropical disease in many regions of the world, particularly sub-Saharan Africa, Asia, Latin America and Papua-New Guinea (Gutierrez et al., 2011). The global crisis of anti-venom supply and the need to distribute antianti-venoms in remote areas of developing countries, where an adequate cold chain cannot be guaranteed, underscore the impor-tance of freeze-dried formulations for snake antivenoms, ensuring their stabilization during transport and storage.
Despite the need to produce more stable and easy to distribute antivenoms, this issue has received little atten-tion by antivenoms manufacturers, and although many commercial formulations are freeze-dried, there is a very limited body of published literature on freeze-drying of snake antivenoms. To the best of our knowledge, the thermal properties of antivenoms and their stability after freeze-drying and during storage in the solid state have not been reported.
The thermal characterization of antivenom formula-tions, and particularly the determination of the glass transition temperature of the maximally freeze-concentrated solution (Tg'), are critical parameters in the development of the freeze-drying cycle (Wang, 2000). Tg' defines the maximally allowable temperature for the pri-mary drying since, if the product temperature exceeds this critical temperature, amorphous collapse could occur (Kasper and Friess, 2011). The stability and activity of freeze-dried antibodies largely depend on the processing conditions and on the use of an adequate stabilizer at the optimum concentration (Sarciaux et al., 1999).
In this work, we performed a thermal characterization of snake antivenoms formulations, assessed the effect of freeze-drying on equine antibodies, and evaluated the effectiveness of sorbitol, sucrose and mannitol as stabilizers in antivenom samples subjected to an accelerated stability test during six months.
2. Materials and methods
2.1. Snake venom
Pools of venoms from adult specimens of the snakes Bothrops asper, Crotalus simus and Lachesis stenophrys, maintained in captivity at the Serpentarium of Instituto Clodomiro Picado, were obtained by mechanical stimula-tion of venom glands, stabilized by freeze-drying and kept at 20 C until use. For neutralization studies, only the
venom ofB. asperwas used, since this species is the most important venomous snake in Central America.
2.2. Antivenoms production and formulation with stabilizers
Plasma from horses immunized with a mixture of the venoms ofB. asper, C. simusandL. stenophryswas used as a starting material. The antivenom immunoglobulins were purified by precipitation with 5% caprylic acid, followed by vigorous agitation for 1 h. Then the immunoglobulins were micro-filtered through an 8
m
m retentive paper (Whatman N 2, Kent, UK), dialyzed against distilled water, and formulated with deionized water at a total protein con-centration of 8 g/dL and a pH of 7.0 (Rojas et al., 1994). Additionally, antivenoms were formulated with either 0.05 M, 0.5 M, 1 M or 2 M sorbitol (Sigma S-7547), 2% or 10% mannitol (Merck-5982), 2% or 5% sucrose (Sigma S-5016) or 0.9% NaCl (Sigma S-1679). Antivenom without excipient was used as a control.2.3. Differential Scanning Calorimetry (DSC)
The glass transition temperature (Tg') was determined using a differential scanning calorimeter, model Q200 (TA Instruments, USA). Twenty mg of each antivenom formu-lation were placed in an aluminum pan that was hermeti-cally sealed and frozen at50C at scan rates of 5e10C/ min, followed by an isotherm of 3 min, and heated at 25C at scan rates of 2.5e5.0C/min. All the Tg' were reported as
the midpoint of the transition.
2.4. Freeze drying microscopy (FDM)
Collapse temperatures were measured using a freezing-drying cryo-stage FDCS 196 (Linkam Scientific Instruments, UK) equipped with a liquid nitrogen cooling system LNP94/ 2 (Linkam Scientific Instruments, UK), a programmable temperature controller, and a vacuum pump Edwards E2M1.5 (Linkam Scientific Instruments, UK). Samples were placed on a 16 mm quartz cover slip and were frozen to
50C at 10C/min. Each sample was heated under vac-uum (about 1 Pa) at 3C/min up to 0C. Direct observation of microscopic collapse was done by using a Nikon Eclipse E600 (Nikon, Japan) polarized microscope with a condenser extension lens.
2.5. Freeze-drying of antivenoms
Ten milliliter vials were filled with 5 mL of each formulation and loaded on a freeze-dryer Benchmark 1100 (Virtis, USA). The samples were frozen at 40 C and annealed at10C for 4 h. The primary drying was con-ducted at20C for 64 h, and the secondary drying at 30C for 4 h and 200 mTorr.
2.6. Residual moisture
106018) and titrated with Karl Fischer reagent (Fisher Sci-entific AL2000-1) until the end point was reached, as determined by the KF processor.
2.7. Reconstitution time
Freeze-dried antivenom samples were reconstituted with 10 mL of 0.85% saline solution (sterile and non-pyrogenic), and the dissolution was observed visually as the vials were gently agitated by hand. The time required to achieve complete dissolution was recorded.
2.8. Turbidity analysis
Turbidity of the preparations was quantified using a turbidimeter model 2020 (La Motte, USA) that was cali-brated with standards (HACH Company, USA) prior to analysis. Turbidity was expressed as nephelometric turbidity units (NTU).
2.9. Neutralization of lethality assay
The neutralization of lethality activity was performed only for B. aspervenom. Mixtures containing a constant amount of this venom, corresponding to four Median Lethal Doses (LD50), i.e. four times the amount of venom required to kill 50% of the animals, and various dilutions of antivenom in 0.12 M NaCl, 0.04 M phosphate, pH 7.2 (PBS), were prepared and incubated for 30 min at 37C. Controls included venom
incubated with PBS instead of antivenom. Aliquots of 0.5 mL of the mixtures were injected, by the intraperitoneal route, to groups offive CD-1 mice (16e18 g). Deaths were recorded during 48 h, and neutralizing activity, expressed as Median Effective Dose (ED50) was estimated by Probits (Solano et al., 2010). The experimental protocols involving animals were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica (Project 82-08) and meet the International Guiding Principles for Biomedical Research Involving Animals (CIOMS, 1986).
2.10. Stability study
The thermal stability of the antivenoms formulated with different excipients was assessed by incubating the sam-ples at 40C±2C and at a relative humidity of 75%±5 during six months. Samples of each formulation were analyzed at the beginning and the end of the study for appearance of the cake, residual moisture content, recon-stitution time, turbidity, electrophoretic pattern and neutralization of the lethal activity ofB. aspervenom.
2.11. Electrophoretic analysis
Twenty
m
g of total protein of each formulation were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, using an acrylamide concentration of 7.5%. Gels were stained with Coomassie Brilliant Blue R-250. The starting voltage was 180 V and gels were destained with a mixture of methanol, ethanol and acetic acid.2.12. Total protein determination
Total protein concentration was determined by a modification of the Biuret test (Parvin et al., 1965) in which 50
m
L of samples (or protein standards) were mixed with 2.5 mL of Biuret reagent and incubated at room tempera-ture during 30 min. Absorbances at 540 nm were recorded and protein concentration was calculated using the equa-tion of the curve obtained by plotting the absorbance of the standards as a function of their protein concentration.2.13. Statistical analysis
Results were expressed as mean±SD from triplicates. The statistical analysis was performed using the software GraphPad InStat™(Goteborg, Sweden). The significance of the differences between the mean values of experimental groups was determined by ANOVA followed by the TukeyeKramer test. Differences were considered statisti-cally significant at values ofP<0.05. Comparison of the ED50 values was performed using the 95% confidence limits. Values were considered significantly different when their 95% confidence limits did not overlap.
3. Results and discussion
3.1. Thermal analysis of frozen formulations
DSC and FDM have been successfully used to determine glass transition and collapse temperatures of therapeutic proteins (Meister and Gieseler, 2009). In this work, the glass transition temperature of the maximally concentrated matrix (Tg') was determined by DSC, as shown inTable 1.
The DSC curve for all frozen formulations showed a slight endothermic shift in heat capacity, associated with glass transition, as well as an endothermic curve close to 0 C corresponding to the melting of ice (data not shown). Moreover, the curve for 10% mannitol formulation also showed an exothermic event in the range of 26 C to
19C, suggesting the further and probable completion of mannitol crystallization during warming. This completion of mannitol crystallization is known as the metastable state. The glass transition temperature for control antivenom, calculated at the midpoint of transition, was 13.5 C, a value close to the Tg' for other pure proteins like myoglobin
Table 1
Glass Transition Temperature (Tg') of frozen snake anti-venom formulations.
Formulation Tg' (C)a
Control 13.5±0.1 Sorbitol 0.05 M 23.6±0.0 Sorbitol 0.5 M 23.0±1.0 Sorbitol 1.0 M 41.3±0.6 Sorbitol 2.0 M 41.4±1.3 Mannitol 2% 19.0±3.0 Mannitol 10% 32.1±0.2 Sucrose 2% 22.9±0.4 Sucrose 5% 22.0±4.0 NaCl 0.9% 22.6±1.4
(10.5 C),
a
-casein (12.5) and ovalbumin (11 C), as reported by Wang (2000). Moreover, the glass transition temperature of the other formulations was lower than the Tg' of control antivenom, and in the formulations where the excipient was the most abundant component, Tg' was close to the value of the pure excipient (Wang, 2000). In anti-venoms formulated with 1 M sorbitol and 2 M sorbitol, the Tg' value was very low; thus, these formulations were analyzed by freeze-drying microscopy to determine the collapse temperature and to confirm the critical tempera-ture of the formulations. On the other hand, a thermal treatment or annealing step was introduced on the freeze-drying cycle to allow complete crystallization of mannitol and removal of the metastable state.3.2. FDM of formulations with sorbitol
The collapse temperature of the antivenoms formulated with 1 M and 2 M sorbitol was determined by FDM (Fig. 1). FDM allows the direct observation of separation between frozen and dried regions of the sample, due to viscousflow of the amorphous phase during heating. This method runs under different experimental conditions to those used in DSC and, therefore, they are considered complementary methodologies in studying thermal properties of protein formulations.
Usually, collapse temperatures are in the range of 2e5C above glass transition temperatures (Pikal and Shah, 1990); however, in this study the collapse temperature did not show a trend, being31C and41C for antivenoms
formulated with 1 M and 2 M sorbitol, respectively. Since collapse temperatures are very low in both samples, the primary drying should be conducted at very low temper-ature and, consequently, the lyophilization process would be very prolonged. Therefore these antivenom formula-tions were not considered suitable for freeze-drying.
3.3. Characterization of freeze-dried formulations
Only the antivenoms formulated with 0.05 M sorbitol, 0.5 M sorbitol, 2% mannitol, 10% mannitol, 2% sucrose, 5% sucrose and 0.9% NaCl were submitted to freeze-drying and further analysis. The visual examination of the freeze-dried
formulations did not show any sign of collapse or shrinkage of the cakes, with the exception of the antivenom formu-lated with 0.9% NaCl. The macroscopic collapse exhibited by this sample was considered unacceptable and this formulation was discarded from further studies. InTable 2, the residual humidity, reconstitution time, turbidity and potency of the freeze-dried antivenoms were compared. All the antivenoms with sorbitol, sucrose or mannitol, except the formulation with 10% mannitol, displayed a lower turbidity than control antivenoms (P<0.05). The effect of polyols for decreasing turbidity of liquid snake antivenoms has already been reported (Segura et al., 2009), as well as the decrease of aggregates in freeze-dried and spray-dried proteins stabilized with sucrose and sorbitol (Maury et al., 2005; Wang et al., 2009).
The reconstitution time of freeze-dried antivenoms is relevant at the clinical setting for two main reasons: (a) because the elapsed time between the snakebite and the beginning of the therapy has an impact on the evolution of the envenomation (Otero et al., 2002) and (b) because difficulty in reconstitution of antivenoms may reflect denaturation of the antibodies, implying loss of stability and neutralizing activity (Theakston et al., 2003). Previous studies have reported pro-longed reconstitution times of 30 and 90 min for some lyophilized antivenoms (Hill et al., 2001; Quan et al., 2010), which might affect the efficacy of treatment, while others recommend different strategies of reconstitution to improve the dissolution time (Gerring et al., 2013). In this work, all the formulations showed reconstitution times lower than 5 min in accordance with times reported for other freeze-dried proteins (Searles et al., 2001; Schersch et al., 2010), corrobo-rating that it is possible to produce antivenoms of easy reconstitution without loss of activity.
The use of sorbitol, mannitol or sucrose in the freeze-drying of antivenoms did not affect the residual moisture content of the samples, since it was lower than 5% for all formulations, with the exception of the control. To the best of our knowledge, the appropriate residual moisture for snake antivenoms has not been reported. It has been sug-gested that lower moisture content leads to more stable protein products (Wang, 2000).
The effect of freeze-drying on the neutralizing potency of antivenom antibodies was assessed by a standardin vivo
test in mice using the intraperitoneal route of injection. For all formulations, the neutralizing potency againstB. asper venom was preserved after freeze-drying. This result sug-gests that antibodies are not affected by the process or by the presence of excipients; however, since the potency assay has a normal variation of 30% (Solano et al., 2010), it is possible that small losses in the potency of the antivenom cannot be detected with this test. Because all formulations maintained their physicochemical and biological properties after freeze-drying, these samples were subjected to a stability study under extreme conditions of temperature and relative humidity during six months.
3.4. Stability study of the freeze-dried formulations
During the development of solid protein preparations, the choice of thefinal formulation is based on the results obtained after the freeze-drying process, but especially on the results generated from stability studies. Accelerated stability studies involve elevated conditions of temperature and humidity, established from the normal conditions of product storage and the climatic zone in which the study is conducted. An important issue about accelerated stability studies is whether the data obtained at high temperatures can be extrapolated to those obtained at real time condi-tions. In this sense some authors have found that, in pro-teins where degradation pathways can be described
separately and the rate-limiting degradation step does not change within a certain temperature range, the prediction of stability based on accelerated studies can provide proper and valuable information (Yoshioka et al., 1994; Mazzobre et al., 1997). However, real-time stability testing should be conducted in parallel with accelerated studies for the selection of the optimalfinal formulation. In this work, the accelerated stability study was conducted with the main objective of identifying the most stable antivenom formu-lation to thermal stress.
Antivenoms formulated with different concentrations of sorbitol, mannitol and sucrose were subjected to an accel-erated stability test at 40C±2C and 75%±5% relative
humidity during six months. Formulations were assessed for appearance of the cake, residual moisture content, reconstitution time, turbidity, electrophoretic pattern and neutralization of the lethal activity against the B. asper venom at the beginning and the end of the study. Analysis of the antivenoms showed that, after six months of incubation at 40 C, the two formulations with sucrose and the
formulation with 2% mannitol decreased their residual hu-midity (P<0.05) as shown inTable 3. This is unexpected, because during the storage of solid proteins, there is usually an increase in residual moisture due to an exchange be-tween the product and the stopper, which is generally steam sterilized (Chang et al., 2005a). The processes involved in the observed decrement of moisture should be further studied. Table 2
Physicochemical and biological characterization of snake antivenom formulations after freeze-drying.
Formulation Reconstitution time (min)a Potency (mg/mL)b Residual moisture (%)a Turbidity (NTU)a,c
Sorbitol 0.05 M 3.0±0.4 2.14 (1.52e3.01) 3.9±0.4 18.3±0.6 Sorbitol 0.5 M 2.9±0.3 1.38 (1.00e1.91) 3.6±0.2 20.0±0.0 Controld 2.9±0.2 1.87 (1.43e2.45) 5.7±1.0 22.0±0.0
Mannitol 2% 0.7±0.3 3.12 (2.33e4.17) 3.1±0.5 22.0±0.0 Mannitol 10% 1.7±0.4 3.39 (2.54e4.53) 2.9±0.3 24.0±0.0 Sucrose 2% 0.7±0.3 2.26 (1.80e2.83) 3.4±0.2 21.0±0.0 Sucrose 5% 1.3±0.4 2.26 (1.69e3.02) 3.5±0.6 21.3±0.6 Controle 2.6±0.3 2.26 (1.69e3.02) 4.0±0.7 24.0±0.0 aResults are presented as mean±S.D. (n¼3).
b Expressed as mg venom neutralized per mL antivenom; 95% confidence intervals are depicted in parenthesis. c NTU: Nephelometric Turbidity Units.
d Control for antivenoms freeze-dried with sorbitol.
eControl for antivenoms freeze-dried with mannitol and sucrose.
Table 3
Physicochemical and biological characterization of freeze-dried antivenom formulations after six months of storage at 40C and 75% relative humidity. Formulation Reconstitution time (min)a Potency (mg/mL)b Residual moisture (%)a Turbidity (NTU)a,c
Sorbitol 0.05 M 10.3±0.3 1.50 (1.20e1.88) 5.4±0.7 110.0±0.0 Sorbitol 0.5 M 11.8±0.5 1.08 (0.79e1.49) NDd 50.0±0.0
Controle 12.6±0.4 1.28 (0.90e1.81) 5.6±0.5 150.0±1.0
Mannitol 2% 5.4±0.3 1.77 (1.41e2.22) 2.4±0.4 78.0±3.0 Mannitol 10% 7.1±0.4 1.92 (1.48e2.49) 1.8±0.4 60.0±0.0 Sucrose 2% 8.8±0.3 1.63 (1.26e2.11) 2.6±0.2 75.0±0.0 Sucrose 5% 5.9±0.2 2.25 (1.68e3.01) 2.7±0.4 55.0±0.0 Controlf 13.4±0.3 2.88 (2.15e3.85) 4.5±0.4 120.0±0.0 aResults are presented as mean±S.D. (n¼3).
b Expressed as mg venom neutralized per mL antivenom; 95% confidence intervals are depicted in parenthesis. c NTU: Nephelometric Turbidity Units.
d Not determined.
eControl for antivenoms freeze-dried with sorbitol.
The impact of moisture content in the storage stability of freeze-dried antibodies has been previously described (Breen et al., 2001; Chang et al., 2005b). It is known that increments in moisture decrease the Tg value, affecting the stability of the formulation (Breen et al., 2001); however, overdrying of proteins is also detrimental for stability. In this study the variation on moisture content during incu-bation was very small and probably does not affect the stability of formulations. Nevertheless, the appropriate re-sidual moisture and its impact in other snake antivenoms cannot be extrapolated from our findings, and must be established for each formulation and for each manufacturer through their own stability studies.
The assessment of reconstitution time after six months of incubation revealed a statistically significant increase in this parameter for all the formulations (P<0.001). Under the conditions of this study, we did notfind any correlation between the excipient content and reconstitution time, and the control antivenom had the highest reconstitution time, whereas the antivenoms freeze-dried with 5% sucrose and 2% mannitol showed the smallest increase in reconstitution time. These results suggest that the presence of mannitol or sucrose exerts a protective effect on the mechanical struc-ture of the cake, preventing the collapse and possibly fa-voring an internal microscopic structure with many pores that allow the hydration of the samples (Overcashier et al., 1999).
The turbidity of antivenoms at the end of stability study showed a significant increase for all the formulations. However, this increment was lower in case of antivenoms freeze-dried with either 0.5 M sorbitol or 5% sucrose, and there was a correlation between turbidity and the con-centration of the excipient. Protein turbidity has been associated with the formation of insoluble aggregates,
which have been associated with the development of adverse events during administration (Cromwell et al., 2006). The protective effect of polyols like sorbitol and mannitol in the development of turbidity in liquid anti-venoms has already been reported (Segura et al., 2009; Rodrigues-Silva et al., 1997, 1999). However, the use of su-crose in the stabilization of liquid or freeze-dried anti-venoms has not been previously described.
The formation of high molecular mass aggregates dur-ing the accelerated stability study was monitored by changes in the electrophoretic profile of antivenoms (Fig. 2). Electrophoretic analysis evidenced the appearance of an intense band at the upper part of the gel after 6 months of incubation at 40±2C, corresponding probably to high molecular mass protein aggregates. This band was not present in the samples with sorbitol at the beginning of the study (Panel A) but it appeared in all formulations with sorbitol, mannitol and sucrose (Panels B and C), but was less intense in the sample formulated with 5% sucrose (Panel C, lane 3). The electrophoretic profiles of antivenoms formulated with mannitol and sucrose at the beginning of the stability test (not shown) were similar to the one described for samples with sorbitol.
These results agree with those reported by other re-searchers, in that sorbitol only slightly protected an IgG1 antibody formulation against aggregation during storage, whereas sucrose improved stability significantly (Chang et al., 2005a). It is postulated that an excipient system that remains at least partially amorphous is necessary for stabilization (Pikal et al., 1991); however, the observation that sorbitol formulations showed poor stability regarding aggregation, demonstrates that an amorphous excipient system is not the only condition necessary for stability of antivenoms.
The properties of the solid protein formulation play an important role in the storage stability. It has been demon-strated that storage of samples above the glass transition temperature of the solid matrix (Tg), might lead to rapid degradation (Chang et al., 2005a; Duddu and Dal Monte, 1997). In this sense, instability of antivenoms formulated with sorbitol and mannitol during storage at 40C could be related to a low glass transition temperature (Tg) for these formulations, probably near or below 40C, which could be directly affecting the physical and chemical stability of the samples. Nevertheless, this issue deserves further investigation.
The effect of the storage on the neutralizing efficacy of antivenoms was assessed by determining the ED50of the formulations. Results showed that, after six months of in-cubation, the neutralizing activity decreased significantly for the two formulations with mannitol.
The stabilizing effect of saccharides and polyols during freeze-drying and the storage of proteins has been explained by two main mechanisms: (a) a thermodynamic stabiliza-tion through direct interacstabiliza-tions between protein and excipient (e.g., hydrogen bonds) that substitute surrounding water molecules (Allison et al., 1999), and (b) a kinetic mechanism that reduces chemical degradation by embed-ding the protein in a glass state of lower molecular mobility (Arakawa et al., 2001). The effectiveness of sorbitol and su-crose to protect the neutralizing activity of antivenoms during six months of storage at 40C can be explained in terms of these mechanisms. Moreover, the decrease in the potency of antivenoms formulated with mannitol might be related to the formation of a crystalline matrix of mannitol, due to the annealing phase carried out during the freeze-drying process. This relationship between the degree of crystallization and the decrease on the protective effect of mannitol in protein formulations has already been reported (Izutsu et al., 1993; Pyne et al., 2003). Our results suggest that the main mechanism involved in the stabilization of freeze-dried snake antivenoms is likely to be the formation of an amorphous matrix instead of the substitution of water, a hypothesis that requires further studies.
The visual examination of the freeze-dried formulations after six months of storage at high temperature showed that all samples retained their cake structure. The mannitol present in formulations probably crystallized completely in the annealing step of the freeze-drying process and, therefore, remained stable during storage. All the anti-venoms, except the sample formulated with 5% sucrose, exhibited a color change to browning with time. This change in the appearance of the cake was more pro-nounced with increased concentrations of mannitol and sorbitol. Reducing sugars, such as glucose and lactose, can react with some residues in proteins to form carbohydrate adducts via the Maillard reaction. This process is also called browning reaction or glycation and has been studied mainly in the food industry (Martins et al., 2001; Burdurlu and Karadeniz, 2003). The browning of solid pharmaceu-tical proteins during storage has been observed for other authors (Li et al., 1996; Wu et al., 1998; Andya et al., 1999), but this phenomenon has not so far been described for freeze-dried antivenoms. Mannitol and sorbitol are not reducing sugars; however impurities present in these
excipients can react with some residues in the proteins (Dubost et al., 1996), and this might explain the observed color change in antivenoms freeze-dried with these poly-ols. A direct relationship between the potency of the anti-venoms and the change in the appearance of the cake was not observed in our experiments. Nevertheless, the role of excipient impurities in promoting drug reactions in the solid state needs to be addressed in future studies since these polyols are widely used in lyophilized pharmaceu-tical forms.
4. Conclusions
Successful freeze-drying of antivenoms requires the conservation of the neutralizing potency of the antibodies as well the improvement of the physicochemical stability of the protein. This study investigated the use of sucrose, mannitol and sorbitol as stabilizers for snake antivenoms formulated as freeze-dried products. DSC provides useful information on the freezing behavior of different formulations and allowed the design of an appropriate freeze drying process. After six months of storage at 40C, all the formulations presented the same residual humidity content, but significant differences were observed in turbidity, reconstitution time and elec-trophoretic pattern. Moreover, all formulations, except antivenoms freeze-dried with mannitol, exhibited the same potency for the neutralization of lethal effect of B. asper venom. The 5% sucrose formulation showed the best stability among the samples tested, while the mannitol and sorbitol formulations underwent significant destabilization and turned brown. Results indicate that sucrose could perform as a better stabilizer than mannitol and sorbitol in the formu-lation of freeze-dried antivenoms. This formuformu-lation may become a good alternative for the production of more stable antivenoms in regions of the world where high temperatures are common and the cold chain is poor.
Ethical statement
This manuscript presents an experimental study per-formed following the standard procedure of scientific ethics.
Acknowledgments
The authors thank our colleagues of Instituto Clodomiro Picado, as well as Sergio Ramírez for his collaboration on DSC analyses. This study was financially supported by Vicerrectoría de Investigacion (Universidad de Costa Rica) project 741-B2-091 and by the program CYTED (project BIOTOX 212RT0467).
Conflicts of interest
The authors declare that there are no conflicts of interest.
Transparency document
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