A protocol for ‘enhanced pepsin digestion’: a step by step method for obtaining pure antibody fragments in high yield from serum

Abstract

The digestion of ovine antiserum under acidic conditions (pH 3.5) by pepsin is highly effective at reducing all unwanted serum components to low molecular weight (≤13 kDa) fragments while leaving the ∼100-kDa F(ab′) 2 intact. The pH is then raised to 6 to stop further digestion and the reaction mixture centrifuged or filtered to remove any insoluble contaminants. Next, unwanted low molecular weight fragments are removed by diafiltration with a 30-kDa nominal molecular weight cut-off membrane leaving an F(ab′) 2 solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. Material of this purity is suitable for many applications but, since all the contaminants are highly acidic, they can be easily removed by passage down an anion-exchange column to yield F(ab′) 2 that is essentially free from pepsin and aggregates with a typical purity of over 96% and yields of 16–19 g/l serum. When an antivenom was processed, ∼78% of the original serum's toxin neutralising capacity was recovered. This simple, high yield protocol for processing serum to highly purified F(ab′) 2 avoids the need for an initial or any subsequent salt precipitation step and can be utilised for either bench or large scale production. If required, a mild reducing agent may be used finally to create Fab fragments.