Refinement strategy for antivenom preparation of high yield and quality.

Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational use of plasma of animal origin. We report on development of an efficient refinement strategy for F(ab')(2)-based antivenom preparation. Process design was driven by the imperative to keep the active principle constantly in solution as a precautionary measure to preserve stability of its conformation (precipitation of active principle or its adsorption to chromatographic stationary phase has been completely avoided). IgG was extracted from hyperimmune horse plasma by 2% (V/V) caprylic acid, depleted from traces of precipitating agent and digested by pepsin. Balance between incomplete IgG fraction breakdown, F(ab')(2) over-digestion and loss of the active principle's protective efficacy was achieved by adjusting pepsin to substrate ratio at the value of 4:300 (w/w), setting pH to 3.2 and incubation period to 1.5 h. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. Developed manufacturing strategy gave 100% pure and aggregate-free F(ab')(2) preparation, as shown by size-exclusion HPLC and confirmed by MS/MS. The overall yield of 75% or higher compares favorably to others so far reported. This optimised procedure looks also promising for large-scale production of therapeutic antivenoms, since high yield of the active drug and fulfillment of the regulatory demand considering purity was achieved. The recovery of the active substance was precisely determined in each purification step enabling accurate estimation of the process cost-effectiveness. Author summary Animal plasma-derived antivenoms constitute the most important therapy against snakebite envenoming. Nowadays this critical treatment has been faced by severe shortage due to low sustainability of current productions, which mostly affects developing countries as those suffering from highest morbidity and mortality rates. Antivenoms' safety and efficacy in clinical setting are highly dependent on manufacturing procedure. Its design should be guided by the tendency to refine immunoglobulin G from residual plasma proteins in only a few easy, simple and efficient purification steps, providing antibody-based product of acceptable physicochemical features and good recovery of protective activity. Here, we developed a compact, feasible and economically viable refinement strategy for antivenom preparation which looks promising for large scale production as well. Process design was driven by the imperative of keeping IgGs or F(ab')(2) fragments constantly in solution in order to preserve stability of their conformations. In each of three main steps-caprylic acid precipitation for removal of contaminants, pepsin digestion of IgGs and chromatographic polishing of F(ab')(2) active principle, optimal performance conditions were defined. As a result, preparation of completely homogenous and aggregate-free final product with over 75% yield was achieved in the most straightforward way. Also, the novel platform has been supported with process efficiency data, so accurate estimation of the cost-effectiveness is enabled.