Streamlined downstream process for efficient and sustainable (Fab')₂ antivenom preparation

Area of Science:

• Biotechnology
• Immunology
• Pharmacology

Background:

• Snakebite envenoming remains a significant health issue with current antivenoms facing limitations in availability, safety, and efficacy.
• Low sustainability of existing antivenom production necessitates technological innovation for higher purity, improved physicochemical characteristics, and cost-effectiveness.

Purpose of the Study:

• To develop a compact, feasible, and economically viable laboratory-scale platform for producing equine F(ab rading_symbol)2 antivenom against Vipera ammodytes ammodytes venom.
• To generate efficiency data for estimating the cost-effectiveness of the developed antivenom production process.

Main Methods:

• Implemented simultaneous caprylic acid precipitation and pepsin digestion in plasma downstream processing.
• Optimized pepsin to substrate ratio (1:30 w/w) and pH (3.2) for controlled IgG breakdown, achieving F(ab rading_symbol)2 formation within 2 hours at 21 °C.
• Utilized diafiltration and flow-through chromatography for final product polishing and assessed in vivo neutralization potency.

Main Results:

• Achieved a high overall yield of 74% through three streamlined steps, meeting regulatory requirements.
• The final F(ab rading_symbol)2 product was free of aggregates and residual pepsin, with minor impurities identified as active IgG/IgM fragments.
• Demonstrated a 3.9-fold increase in specific activity compared to the initial plasma.

Conclusions:

• Engineered a highly streamlined method for equine F(ab rading_symbol)2 antivenom production.
• The process is simple, rapid, and achieves high yield and regulatory compliance, suitable for laboratory settings.
• The platform shows promise for large-scale manufacturing of effective and potentially more affordable antivenoms.