Abstract
Parenterally administered snake antivenom immunoglobulins are the only specific treatment for envenoming by snakebites. Endotoxin removal is a necessary part of good manufacturing practice for antivenom products to avoid life-threatening consequences associated with injecting endotoxin-contaminated product. Optimization of pH is an essential factor in endotoxin purification. This study aimed to compare ultrafiltration, ion-exchange chromatography and affinity resin-based chromatography techniques at different pH values to select the depyrogenation method with the highest endotoxin removal efficiency and optimum protein/product recovery. Affinity resin-based chromatography achieved 91.2% protein recovery at acidic pH without detectable endotoxins, while ion-exchange chromatography achieved 74.42% protein recovery at pH 7.5. In contrast, ultrafiltration achieved the lowest protein recovery compared to other chromatography techniques. In addition, ultrafiltration was ineffective in removing serum albumin (~ 42-57 kDa) and low molecular weight (MW) Fc fragments (~ 24-31 kDa). In conclusion, affinity resin-based chromatography has proven to be the most effective endotoxin removal method, while ultrafiltration may not be appropriate for the removal of bacterial LPS from antivenom sera. Moreover, this study demonstrated the existence of an optimum pH for each chromatographic method for the purpose of producing sterile and endotoxin-free snake antivenoms.