Freeze-dried snake antivenoms formulated with sorbitol, sucrose or mannitol: Comparison of their stability in an accelerated test

Abstract

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 -13.5 °C and -41 °C. 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 40 °C, all the formulations presented the same residual humidity, but significant differences were observed in turbidity, reconstitution time and electrophoretic pattern. Moreover, all formulations, except antivenoms freeze-dried with mannitol, exhibited the same potency for the neutralization of lethal effect of Bothrops asper venom. 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.

Safety and efficacy of a freeze-dried trivalent antivenom for snakebites in the Brazilian Amazon: An open randomized controlled phase IIb clinical trial

“…A main concern is the conservation of the freeze-dried AV pharmacokinetic properties and further neutralizing potency of the antibodies submitted to lyophilization [ 25 ]. Although positive results from thermal characterization of the effect of freeze-drying on equine antibodies and the effectiveness of stabilizers in freeze-dried AVs have been reported [ 18 , 19 ], to the best of our knowledge this is the first clinical trial aiming to compare the safety and efficacy of a freeze-dried AV with a liquid formulation. This study did not include data on the possible increase in the cost of AV production due to the freeze-drying process.…”

“…The protective effect of polyols like sorbitol and mannitol in the development of turbidity in liquid AVs has already been reported [ 48 – 50 ]. More recently, the use of sucrose in the stabilization of liquid or freeze-dried AVs has been described as a better stabilizer than mannitol and sorbitol in the FDTAV formulation [ 18 , 19 ].…”

“…This additional step and addition of different stabilizers lead to AVs with a higher stability compared to liquid formulations, especially in tropical regions where high temperatures could affect the activity of immunoglobulins [ 18 – 20 ]. Physicochemical characterization studies of commercial freeze-dried AVs from India, Mexico, Thailand and Costa Rica showing good quality standards are available in the literature [ 18 , 20 ]. However, knowledge about freeze-dried AVs efficacy and safety obtained from clinical trials is still very limited.…”

Venom and Antivenom of the Redback Spider (<i>Latrodectus hasseltii</i>) in Japan. Part II. Experimental Production of Equine Antivenom against the Redback Spider

“…Further studies are warranted to improve the manufacturing process to provide better yield, considering recent reports, describing a new non-mineral adjuvant that can minimize burden on the horses (24,25) and describing a stabilizer for freeze-drying of antivenom (26).…”

Lachesis stenophrys venom reduces the equine antibody response towards Bothrops asper venom used as co-immunogen in the production of polyspecific snake antivenom

“…Antivenom immunoglobulins were purified by the caprylic acid method (Rojas et al, 1994) and anti-B, anti-L and anti-BL antivenoms were formulated at 4.5 g/dL total protein, 0.9 g/dL NaCl, 0.25 g/dL phenol and pH 7.0. All antivenoms were freezedried without stabilizers following the procedure described by Herrera et al (2014a), and stored at room temperature (20e25 C) until use.…”