Comprehensive LC–MS/MS Label-Free Profiling of Antivenoms for Detecting Low-Abundance Proteins and Process Evaluation

Snakebite envenoming, recognized as a Neglected Tropical Disease since 2017, continues as major public health challenge in tropical and subtropical developing countries. Heterologous antivenom remains the only proven effective treatment, and in Brazil, public laboratories manufacture it exclusively under strict regulation. Since 2017, the Brazilian Health Regulatory Agency (ANVISA) has required more rigorous antivenom characterization, particularly regarding contaminants and impurities. However, the classical biochemical and immunochemical assays recommended by WHO Guidelines are primarily qualitative or semi-quantitative, and their limited sensitivity impairs the detection of low-abundance proteins, compromising the full resolution of antivenom complexity. In order to address this gap, we applied an integrated proteomic workflow to characterize the protein composition of hyperimmune heterologous sera. Bothrops antivenoms obtained from simulations of three distinct manufacturing processes were analyzed by size-exclusion liquid chromatography (SEC), SDS-PAGE and two-dimensional electrophoresis, western blot and label-free LC–MS/MS. The last technique and SDS-PAGE also were used to analyze the SEC fractions from the antivenoms. Chromatographic, electrophoretic, immunoreactivity and LC–MS/MS profiles were complementary and indicated both the active components and impurities. Moreover, the label-free approach provided greater proteome depth, enabling the identification and relative quantification of low-abundance species, while enrichment of SEC fractions from the antivenom, demonstrated by electrophoresis and proteomic analysis, revealed proteins not detected in unfractionated samples. The proposed strategy enabled more comprehensive characterization of the antivenoms and demonstrated differences among sera produced by distinct processes, highlighting its potential to evaluate the impact of manufacturing changes on product quality. Furthermore, this approach applies to quality control, represents an advancement over the analytical methodologies described in the WHO Guidelines, and can be broadly applied to antivenoms and other biological products.