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.

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Measles virus and mumps virus (MeV and MuV) are enveloped RNA viruses used for production of live attenuated vaccines for prophylaxis of measles and mumps disease, respectively. For biotechnological production of and basic research on these viruses, the preparation of highly purified and infectious viruses is a prerequisite, and to meet that aim, knowledge of their stability and biophysical properties is crucial. Our goal was to carry out a detailed investigation of the stability of MeV and MuV under various pH, temperature, shear stress, filtration and storage conditions, as well as to evaluate two commonly used purification techniques, ultracentrifugation and diafiltration, with regard to their efficiency and effect on virus properties. Virus titers were estimated by CCID50 assay, particle size and concentration were measured by Nanoparticle tracking analysis (NTA) measurements, and the host cell protein content was determined by ELISA. The results demonstrated the stability of MuV and MeV at pH <9 and above pH 4 and 5, respectively, and aggregation was observed at pH >9. Storage without stabilizer did not result in structural changes, but the reduction in infectivity after 24 hours was significant at +37 °C. Vortexing of the viruses resulted in significant particle degradation, leading to lower virus titers, whereas pipetting had much less impact on virus viability. Diafiltration resulted in higher recovery of both total and infectious virus particles than ultracentrifugation. These results provide important data for research on all upstream and downstream processes on these two viruses regarding biotechnological production and basic research.

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