Development of a venom-based affinity chromatography matrix for the selective enrichment of F(ab')₂ fragments from crotalic antivenom.

Abstract

Snake antivenoms remain the only effective treatment for envenomation; however, current manufacturing practices often result in formulations containing substantial amounts of non-specific serum proteins and heterologous immunoglobulins, which contribute to adverse reactions and limit therapeutic performance. Here, we developed and evaluated a venom-based affinity chromatography platform in which Crotalus durissus terrificus toxins were covalently immobilized on CNBr-activated Sepharose 4B to enable selective isolation of F(ab')₂ fragments (BF) from crotalic antivenom (CAv). Ten chromatographic runs were conducted across two experimental sets to assess method repeatability and intermediate precision. Chromatographic profiles were highly consistent across all assays, with intra-run and inter-run relative standard deviations (RSD) ≤ 15%, confirming the robustness of the purification method. Quantitative proteomics demonstrated substantial enrichment of BF and a marked depletion of non-specific serum proteins such as albumin, fibrinogen, fibronectins, and serotransferrins. ELISA assays revealed an 8-fold increase in BF by the enriched fraction relative to the CAv. In vitro functional assays demonstrated that BF exhibited greater inhibition of enzymatic activity than CAv across the tested time points. Together, these results demonstrate the high selectivity and reliability of the venom-based affinity matrix and may support its integration as an incremental bioprocessing step to enhance the purity, quality, and safety of antivenoms. This platform demonstrates a proof of concept for advancing next-generation crotalic antivenom production.

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