Single-reagent one-step procedures for the purification of ovine IgG, F(ab′)2 and Fab antivenoms by caprylic acid

• Caprylic acid fractionation is a valuable tool for purifying IgG antibodies.
• Subsequent addition of pepsin or papain produces F(ab′)2 or Fab antibody fragments
• Here we describe ovine antivenom produced using new one-step single-reagent methods.
• These methods result in a substantial reduction in processing time.
• Resulting ovine F(ab′)2 and Fab antivenoms are comparable to traditional antivenom.

Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab′)2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab′)2 and Fab fragments, respectively. A “single-reagent” method was developed for the production of F(ab′)2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4 h at 37 °C. For ovine Fab antivenom production from whole antiserum, the “single reagent” comprised of caprylic acid, papain and l-cysteine; after incubation at 37 °C for 18–20 h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab′)2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate.