A Heparin Purification Process Removes Spiked Transmissible Spongiform Encephalopathy Agent

BSTRACT In 2000, bovine heparin was withdrawn from the US market for fear of contamination with bovine spongiform encephalopathy (BSE) agent, the cause of variant Creutzfeldt-Jakob disease in humans. Thus, US heparin is currently sourced only from pig intestines. Availability of alternative sources of crude heparin, a life-saving drug, would benefit public health. Bovine heparin is an obvious option, but BSE clearance by the bovine heparin manufacturing process should be evaluated. To this end, using hamster 263K scrapie as a surrogate for BSE agent, we applied a four-step bench-scale heparin purification protocol resembling a typical heparin manufacturing process to investigate removal of the spiked scrapie agent. We removed aliquots from each step and analyzed them for residual abnormal prion protein (PrP TSE ) using a sensitive in vitro method, real-time quaking-induced conversion (RT-QuIC) assay, and for infectivity using animal bioassays. The purification process reduced infectivity by 3.6 log 10 and removed PrP TSE , measured as seeding activity, by 3.4 log 10 . NaOH treatment was the most effective removal step tested. We also investigated NaOH at different concentrations and pH: the results showed that as much as 5.2 log 10 of PrP TSE seeding activity was removed at pH 12.5. Thus, changes to the concentration, treatment time, and temperature of alkaline extraction might further improve removal. Our results, using a basic heparin manufacturing process, inform efforts to reintroduce safe bovine heparin in the USA.