Prion depletion and preservation of biological activity by preparative chaotrope ultracentrifugation.

摘要

Prions are characterized by unusual physicochemical properties, such as insolubility and resistance to proteases, and maintain infectivity after contact with disinfectants and decontamination procedures active against conventional pathogens. To date, most methods for prion inactivation are either incomplete or unacceptably harsh for the purification of fragile biotherapeutics. Here we describe a simple prion removal procedure that takes advantage of differential sedimentation and denaturation of prions. Prion-spiked fluids were layered onto an intermediate sucrose cushion and an 8M urea solution, and subjected to single-step ultracentrifugation. Due to their insolubility, prions rapidly traveled through the sucrose cushion into the urea solution. Prion infectivity in the upper phase was reduced by at least 3.2 logs, or up to 6 logs or more. Very little soluble protein was lost from the input sample and a proof-of-principle experiment demonstrated only marginally reduced biological activity of spiked enzyme after ultracentrifugation. This procedure is likely to synergize with nanofiltration and other prion removal steps in the treatment of batches of raw and semifinal biopharmaceutical materials.