title: A Heparin Purification Process Removes Spiked Transmissible Spongiform Encephalopathy Agent.
abstract: In 2000, bovine heparin was withdrawn from the US market for fear of contamination with bovine spongiform encephalopathy (BSE) agent, the cause of variant Creutzfeldt-Jakob disease in humans.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 (PrPTSE) 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 log10and removed PrPTSE, measured as seeding activity, by 3.4 log10. 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 log10of PrPTSEseeding 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.
authors.affiliation: Food and Drug Administration, 10903 New Hampshire Avenue, Building 52/72, Room 4336, Silver Spring, Maryland, 20993, USA.
authors.forename: Cyrus
authors.initials: C
authors.lastname: Bett
authors.affiliation: Food and Drug Administration, 10903 New Hampshire Avenue, Building 52/72, Room 4336, Silver Spring, Maryland, 20993, USA.
authors.forename: Ksenija
authors.initials: K
authors.lastname: Grgac
authors.affiliation: Food and Drug Administration, St Louis, Missouri, USA.
authors.forename: Dianna
authors.initials: D
authors.lastname: Long
authors.affiliation: Food and Drug Administration, St Louis, Missouri, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Karfunkle
authors.affiliation: Food and Drug Administration, St Louis, Missouri, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Keire
authors.affiliation: Food and Drug Administration, 10903 New Hampshire Avenue, Building 52/72, Room 4336, Silver Spring, Maryland, 20993, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Asher
authors.affiliation: Food and Drug Administration, 10903 New Hampshire Avenue, Building 52/72, Room 4336, Silver Spring, Maryland, 20993, USA. Luisa.gregori@fda.hhs.gov.
authors.forename: Luisa
authors.initials: L
authors.lastname: Gregori
mesh_terms: D000818:Animals; D002417:Cattle; D016643:Encephalopathy, Bovine Spongiform / Q000517:prevention & control* / Q000635:transmission; D006493:Heparin / Q000302:isolation & purification*; D007422:Intestines / Q000737:chemistry; D008647:Mesocricetus; D000072002:Prion Proteins / Q000302:isolation & purification*; D012608:Scrapie / Q000635:transmission; D012972:Sodium Hydroxide; D014020:Tissue Extracts
chemical_list: D000072002:Prion Proteins; D014020:Tissue Extracts; D012972:Sodium Hydroxide; D006493:Heparin
keywords: RT-QuIC; bioassay; heparin; prion; scrapie; transmissible spongiform encephalopathy (TSE)