Effect of pepsin digestion on the antivenom activity of equine immunoglobulins

Enzyme digestion of animal-derived sera followed by antibody purification is a classical process used to prepare snake antivenoms worldwide. In this work, we have studied the effect of the harsh conditions prevailing during the digestion step on the activity of the final product, F(ab′)₂. To this purpose, the recovery of the activity of anti-Bothrops hyperimmune equine plasma was determined after pepsin digestion under different sets of processing conditions. The balance between pH level and reaction time was found to be critical, reflecting a compromise between complete cleavage of immunoglobulins and strong denaturation of the F(ab′)₂ fragments. For pH in the range 2.8–3.2, 30–65% of the initial activity was lost depending mainly on the processing time, as determined by a competition ELISA technique. Pepsin digestion was also carried out with purified immunoglobulins from the same plasma. SDS PAGE run on the digested immunoglobulins allowed us to verify that the lightest isotypes were more resistant to digestion than the heavier ones. In conclusion, for equine F(ab′)₂ antivenom production, it seems convenient to carry out digestion at pH values sufficiently low to ensure that total IgG breakdown is achieved in the shortest time compatible with precise operation in the production scale.