DNA clearance in chromatography of proteins, exemplified by affinity chromatography.

ABSTRACT

Complications of DNA clearance in protein chromatography using the conventional methodology of spiking experiments are reported. Protein A affinity chromatography demonstrated this complications in a small scale experiment. A concentrated hybridoma culture supernatant was spiked with DNA extracted from hybridoma cells fed with [3H]thymidine. Protein A affinity chromatography was subsequently carried out. The column effluent was collected in fractions, and each fraction was analyzed for radioactivity and IgG levels. A substantial amount of DNA was eluted before the main IgG peak. Frequently a small peak is observed in front of the main peak in protein chromatography. This phenomenon can be explained by either displacement effects, or incomplete washing, or hysteresis during the adsorption and desorption conditions. Fractionation at the beginning of elution is critical to the maintenance of a high standard protein purity.