engleski

Equine F(ab')2-based antivenom preparation by simultaneous caprylic acid fractionation and pepsin digestion

Antivenoms obtained from hyperimmune animal plasma have been the only specific and effective therapy against snakebites. Design of the ideal process preceding their commercial scale production should be guided by the tendency to refine IgGs or their antigen- binding fragments from residual plasma proteins in the most straightforward way, providing final product of retained potency and reduced side-effects inducing potential. Here we report simple, feasible and economically viable downstream processing strategy for Vipera ammodytes-specific antivenom preparation that has been guided by the imperative of constant maintenance of F(ab’)2 fragments in solution, as a precautionary measure against their conformational or structural change due to precipitation or binding to chromatographic support. Thus, protection of the active principle from possible degradation and/or aggregation was ensured. Also, for the first time caprylic acid fractionation of horse plasma was successfully co- performed with the pepsin digestion step. Developed mode employing simultaneous precipitation of contaminating proteins together with Fc fragment removal from IgGs resulted in F(ab’)2- based product that was additionally polished by anion-exchange chromatography in the flow-through mode, under conditions that prefer binding of processing by- product traces, especially pepsin. Such compressed manufacturing procedure yielded over 70% of active principle, as properly and reliably estimated by several in vitro methods. The final preparation was distinguished by high purity and preserved in vivo neutralisation efficacy in comparison to the starting plasma pool.

Antitoxin downstream processing ; Hyperimmune plasma ; Antivenom ; IgG ; F(ab')2 ; Caprylic acid ; Pepsin digestion ; Ion-exchange chromatography

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