Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography

Abstract

A combined process of caprylic acid (CA) precipitation and ion-exchange chromatography on SP-Sepharose was studied as a means to fractionate pepsin-digested horse antivenom F(ab′) 2 antibody. In the CA precipitation, the optimal concentration for fractionation of F(ab′) 2 from pepsin-digested horse plasma was 2%, in which 89.61% of F(ab′) 2 antibody activity was recovered in the supernatant with 1.5-fold purification. A significant amount of pepsin was not precipitated and remained active under these conditions. An analytical cation exchanger Protein-Pak SP 8HR HPLC column was tested to establish optimal conditions for the effective separation of IgG, albumin, pepsin and CA from the F(ab′) 2 product. From these results, the supernatant from CA precipitation of pepsin-digested plasma was subjected to a SP-Sepharose column chromatography using a linear salt gradient. With stepwise elution, a peak containing F(ab′) 2 antibody could be obtained by elution with 0.25 M NaCl. The total recovery of antibody was 65.56% with 2.91-fold purification, which was higher than that achieved by ammonium sulfate precipitation. This process simultaneously and effectively removed residual pepsin, high molecular weight aggregates and CA in the final F(ab′) 2 product, and should be suitable for large-scale fractionation of therapeutic equine antivenoms.