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′)2antibody. In the CA precipitation, the optimal concentration for fractionation of F(ab′)2from pepsin-digested horse plasma was 2%, in which 89.61% of F(ab′)2antibody 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′)2product. 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′)2antibody 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′)2product, and should be suitable for large-scale fractionation of therapeutic equine antivenoms. © 2003 Elsevier B.V. All rights reserved.