Abstract
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.
Highlights
MethodsAdult mice (strain NIH Ola/Hsd, both sex, 18–20 g) for lethal toxicity neutralisation assay were purchased from the Institute of Immunology Inc
Antivenoms prepared from hyperimmune animal plasma, mostly equine or ovine, are the only specific therapeutics for rapid counteracting post-snakebite pathophysiological manifestations
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
Summary
Adult mice (strain NIH Ola/Hsd, both sex, 18–20 g) for lethal toxicity neutralisation assay were purchased from the Institute of Immunology Inc. Animal use protocols were established in accordance to Croatian Law on Animal Welfare (2017) which strictly complies with EC Directive (2010/63/EU). Experiments involving animals were approved by the Croatian Ministry of Agriculture, Veterinary and Food Safety Directorate The approval is based on the positive oppinion of the National Ethical Commitee (EP 110/2017). During the test period mice were housed in a 12-h light/12-h dark cycle and at a constant temperature of 22 ̊C. A standard mouse diet (Mucedola srl., Italy) and water were supplied ad libitum. Animal monitoring for signs of pain, suffering and distress associated with procedure was performed following severity assessment protocol
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