DNA CLEARANCE IN CHROMATOGRAPHY OF PROTEINS, EXEMPLIFIED BY AFFINITY-CHROMATOGRAPHY

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