Heparin is a widely used injectable anticoagulant. In the 1990s, bovine-derived heparin was withdrawn from the US market because of concerns about possible contamination of bovine tissues with the agent of bovine spongiform encephalopathy (BSE), the causative agent of variant Creutzfeldt-Jakob disease (vCJD) in humans (1). Currently, only porcine heparin, mostly from China, is marketed in the United States. The US Food and Drug Administration has encouraged reintroduction of bovine-sourced heparin into the US market to improve the reliability of the heparin supply chain by diversifying sources (2,3). The risk that BSE agent might contaminate bovine tissues is now very small because of safeguards implemented during the BSE crisis (4).

We previously showed that a model 4-step bench-scale heparin manufacturing process cleared substantial amounts of spiked scrapie agent, a surrogate for BSE agent (5). Our protocol yielded heparin with physicochemical identity, purity, and potency similar to those of United States Pharmacopeia (USP) standard heparin. In this study, we spiked commercial crude bovine heparin with BSE agent itself and processed samples using the same manufacturing process we applied to scrapie agent. We tested each intermediate product for residual abnormal prion protein (PrP^TSE, a biochemical marker of BSE) and infectivity. We assayed BSE infectivity using intracerebral inoculations of 30-μL volumes into BSE-susceptible transgenic mice (TgBo110) overexpressing the bovine prion-protein–encoding (PRNP) gene (6). To overcome heparin’s acute toxicity when administered intracerebrally into mice, we diluted the samples; 10⁻⁴ was the lowest dilution tolerated.

We ended the study 2 years after inoculations, testing brains of all mice for PrP^TSE using the HerdCheck BSE-Scrapie Ag Test (IDEXX Laboratories, https://www.idexx.com) (7), which was previously found to be more sensitive than Western blots (8), to assign final disease status (Table). We detected infectivity in samples up to the diatomaceous-earth (DE) filtration step. We estimated removals by DE filtration conservatively, assuming that a 10-fold lower dilution, not tested, would have infected all mice. Sodium hydroxide (NaOH) treatment removed 1.7 log₁₀ of BSE infectivity and DE filtration removed >1.1 log₁₀ of BSE infectivity. To increase sensitivity of the mouse bioassay, we removed heparin by centrifuging samples (20,000 × g, 1 hr, 4°C), washed the pellets, resuspended them in inoculation buffer, and inoculated mice as described. We tested brains of all mice for PrP^TSE as reported previously (5). We detected residual infectivity in all aliquots, including the final product. NaOH treatment removed 1.5 log₁₀ of BSE infectivity. We estimated removals by other steps. DE filtration removed ≥1.4 log₁₀ of BSE infectivity. The hydrogen peroxide bleaching and methanol precipitation (final product) steps each removed <1 log₁₀ of infectivity, considered negligible. Thus, cumulatively, scaled-down heparin purification removed a total of >2.9 log₁₀ of BSE infectivity; NaOH treatment and DE filtration were the only effective steps.

We also quantified residual PrP^TSE in each sample using the real-time quaking-induced conversion (RT-QuIC) assay with hamster–sheep chimeric prion protein (9) as substrate, expressing results as log₁₀ 50% seeding doses (SD₅₀), as reported previously (5). We detected PrP^TSE in unspun BSE spike and NaOH-treated samples but only inconsistent signals in aliquots from successive steps (data not shown). To increase sensitivity and remove heparin interfering with RT-QuIC at low concentrations of PrP^TSE, we centrifuged all samples as we did previously. To quantify PrP^TSE, we resuspended pellets and serially diluted each sample in phosphate-buffered saline 0.05% sodium dodecyl sulfate, adding 2 µL of each dilution to seed RT-QuIC, each dilution into quadruplicate wells (see log₁₀ SD₅₀ values in Table). NaOH treatment removed 2.4 log₁₀ of PrP^TSE and DE filtration steps removed 1.3 log₁₀ of PrP^TSE. Hydrogen peroxide bleaching and methanol precipitation reduced PrP^TSE by only negligible amounts. Thus, processing from crude heparin to final pharmaceutical heparin cumulatively removed 3.7 log₁₀ of spiked PrP^TSE.

We showed previously, using a rodent-adapted scrapie agent, that heparin processing removed 3.6 log₁₀ of scrapie infectivity and 3.4 log₁₀ of PrP^TSE (5). Here, we report studies with the more relevant BSE agent itself, showing similar reduction by 3.7 log₁₀ of PrP^TSE. We could demonstrate only >2.9 log₁₀ reduction in infectivity, because the starting titer of the BSE-infected brain homogenate was low. However, we detected both residual BSE infectivity and PrP^TSE seeding activity after final steps of processing, so our model process did not yield sterile heparin. We found NaOH treatment and DE filtration to be the most effective steps for removing both BSE infectivity and PrP^TSE seeding activity, consistent with previous results using scrapie agent.

Overall, our data suggest that typical heparin manufacturing is likely to remove substantial amounts of BSE agent. Furthermore, a probabilistic model assessing the vCJD risk for bovine heparin sourced from cattle in the United States and Canada estimated the risk to be very low (10). The demonstrated ability of a typical heparin purification process to remove substantial amounts of contaminating BSE agent, taken together with careful selection of low-risk bovine material to manufacture heparin, provides additional assurance of safety, supporting eventual reintroduction of bovine heparin to the US market.

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