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 a a Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jos e, Costa Rica b Department 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 c Laboratorio Nacional de Nanotecnología, LANOTEC-CENAT, San Jos e, Costa Rica article info 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 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 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 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. © 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 chemically 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: maria.herrera_v@ucr.ac.cr, mariaherrv@hotmail. com (M. Herrera). Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon http://dx.doi.org/10.1016/j.toxicon.2014.07.015 0041-0101/© 2014 Elsevier Ltd. All rights reserved. Toxicon 90 (2014) 56e63