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A Heparin Purification Process Removes Spiked Transmissible Spongiform Encephalopathy Agent

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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In 2000, bovine heparin was withdrawn from the US market for fear of contamination with bovine spongiform encephalopathy (BSE) agent, the cause of variant Creutzfeldt-Jakob disease in humans.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 log10 and 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 log10 of PrPTSE 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.

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Acknowledgements

We thank members of our laboratories and especially Teresa Pilant, Dianne McMahon, and Oksana Yakovleva and the staff in the FDA Division of Veterinary Services for the outstanding care of animals. We are in special debt to the staff in the laboratory of Byron Caughey at Rocky Mountain Laboratory, NIAID, NIH, Hamilton, Montana, for sharing the recombinant constructs and their knowledge of RT-QuIC.

The animal work was approved by the FDA Animal Care and Use Committee (ASP#2014-14).

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Correspondence to Luisa Gregori.

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This work was supported by intramural grants from the US Food and Drug Administration.

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All authors declare no conflict of interest.

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Bett, C., Grgac, K., Long, D. et al. A Heparin Purification Process Removes Spiked Transmissible Spongiform Encephalopathy Agent. AAPS J 19, 765–771 (2017). https://doi.org/10.1208/s12248-017-0047-y

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