Thermal stability of antivenoms: evaluation of protein aggregates formation

Abstract

Due to its high impact, envenomation by venomous animals has been considered a neglected tropical disease by the World Health Organization (WHO), and the antivenoms are included in the WHO List of Essential Medicines, making serum therapy the mainstay for the improvement of patients. Serum therapy involves the transfer of immunoglobulins from an immunized individual to an unimmunized one, in order to provide rapid protection against a particular antigen. In Brazil, antivenoms of heterologous origin containing F (ab ') 2 fragments are used, because, according to literature data, intact IgGs and the Fc portion are shown to be able to induce the generation of anaphylatoxins (C3a, C4a, C5a). In previous studies, our group demonstrated that many antivenoms contain in the packaged product protein aggregates and IgG molecules that have been resistant to purification methods, as well as contaminants that are not targeted to venom antigens and do not participate in its therapeutic effect . All these factors may contribute to the appearance of adverse reactions in patients submitted to serum therapy. Such reactions have been attributed to complement activation, mainly by the presence of aggregates. In previous studies of our group, it was suggested the use of complement activation methods as parameters to confirm the presence of protein aggregates in the antivenoms and to define acceptable levels of these aggregates. The possibility of protein aggregation during the storage of the packaged antivenoms or of the hyperimmune plasmas that constitute the raw material for the production of antivenoms can not be ruled out, even if they are within the validity periods and at the appropriate temperature. Infrastructure conditions for transport and storage should also be considered in order to determine the effect of time and temperature on the generation of these aggregates. The present project aims to study the impact of time and storage temperature of hyperimmune plasmas and packed antivenoms on the formation of protein aggregates, which could increase the incidence of hypersensitivity reactions. Packaged antivenom samples were previously obtained and incubated for 30, 60 and 90 days at temperatures of 25 ° C and 37 ° C. After the incubation periods, the samples were frozen at -20 ° C for 48 months. Samples from the same batches were maintained at 4 ° C for the entire period, for comparison purposes. Hyperimmune plasma samples were also obtained and will be incubated at 37 ° C for 30, 60 and 90 days, compared to the same samples kept at 4 ° C. All samples will be evaluated for protein concentration, electrophoretic profile and Western Blot reaction for IgG detection, allowing the visualization of possible protein aggregates, as well as intact immunoglobulins, contaminating proteins or degradation products. In addition, activation of the complement system by the samples will be evaluated by the generation of anaphylatoxins in vitro, by incubating the samples with normal human serum, and analyzing the generation of C3a, C4a and C5a by flow cytometry. The results will allow to determine the impact of time and temperature on the formation of aggregates in antivenoms, contributing to the development of better quality antivenoms, and also to the possible incorporation of new methods of quality control of antivenoms.