Sanitization of Equipment and Material Used in the Production of Plasma Derivatives by Standard Procedures Results in Removal and Inactivation of Prions.

Prions are novel infectious agents causing neurodegenerative disorders such as scrapie, bovine spongiform encephalopathy (BSE), and (variant) Creutzfeldt-Jakob disease ((v)CJD). The infectious agents consist mainly, if not exclusively, of a malformed protein termed PrPSc, which accumulates in the brain of infected individuals. This PrPSc is resistant to proteinase K treatment. As there are concerns that minute amounts of prions from a donor in the incubation period for CJD / vCJD may contaminate the equipment and material used for the production of plasma-derived medicinal products and, therefore, may taint subsequently produced batches of these products, we evaluated the cleaning capacity of selected cleaning procedures used within the production of plasma proteins: NaOH and commercial alkaline cleaning solutions at different concentrations to clean and sanitize equipment and materials (e.g., resin). Sanitization of equipment was evaluated employing microsomal preparations from brains of Sc237-infected hamsters dried onto stainless steel coupons for 1 day, subsequently placed in 0.1 N NaOH, various concentrations of commercial cleaning solutions CIP-100 / CIP-150, and water as a control, respectively, followed by rinsing in water, and recovered from the coupons by wiping off with swabs. The cleaning efficacy was calculated as the difference of the recovered prion load (in log10) of a coupon with dried-on PrPSc without any further treatment and after treatment with 0.1 N NaOH or commercial cleaning solutions. PrPSc was quantified utilizing a Conformation Dependent Immunoassay (CDI) [Safar et al., Nat Med1998; 4: 1157–1165] in a sandwich configuration. In addition, prion inactivation by NaOH and commercial alkaline cleaning solutions could be demonstrated when prions were incubated in these solutions and treated with proteinase K. Furthermore, the removal of prions from chromatographic resins was studied by incubation of resin with microsomes from Sc237 hamster brain, extensive washing of the resin and subsequently incubating the resin in either water for injection or in 0.1 N NaOH followed by a treatment of both samples with or without proteinase K (PK). In order to extract all residual prion protein from the resin, all four samples were incubated at elevated temperature in guanidinium-HCl and residual prions were quantified utilizing a sandwich ELISA format according to the CDI. From the PK treated sanitized resin no residual prion protein could be detected resulting in a reduction factor of ≥3 log10 compared to non-sanitized (WFI incubated) resin. The experimental data clearly demonstrate that prions, if they were in plasma, would be removed from the surface of equipment and resins used in the production of plasma-derived products and furthermore inactivated by standard cleaning procedures according to cGMP. Therefore, a potential risk for batches of plasma-derived medicinal products produced in succession due to cross-contamination by carry-over of prions can be excluded.