Validation of TSE Agent Removal by Immunoaffinity and Ion Exchange Chromatography Steps of the ReFacto Manufacturing Process.

Abstract

ReFacto® (moroctocog alfa), a recombinant Factor VIII approved for treatment of hemophilia A is produced by a mammalian cell culture process that includes therapeutic-grade human serum albumin (HSA) in the cell culture medium. The plasma-derived HSA used in the cell culture process is sourced from countries whose blood supply does not pose a significant transmissible spongiform encephalopathy (TSE) risk, and is manufactured utilizing a fractionation process that has demonstrated the capability to significantly reduce the level of experimentally added TSE infectivity. While to date, no case of TSE has been identified with a history of exposure to fractionated HSA, Wyeth has conducted a study to demonstrate that the ReFacto manufacturing process has significant capacity to remove a TSE agent if it were present in the HSA. The ReFacto purification process consists of five chromatographic steps, two of which were validated for removal of a TSE agent: the immunoaffinity step (8A4 Sepharose chromatography) and anion exchange step (Q Sepharose chromatography). The GLP study was performed using appropriately qualified lab-scale systems, which model the performance of the production equipment. Brain homogenates from scrapie-infected hamsters (263K strain) were added to loads of the respective chromatography steps and levels of the scrapie agent in the product pools were determined using a validated Western blot quantitation method. Duplicate runs were performed to assess the consistency of the TSE agent reduction. The results show that both chromatography steps provide significant clearance of the TSE agent, with greater than a billion-fold (9 log) reduction in infectivity provided by the two process steps in combination. The consistency of the duplicate run results was very good (less than 0.7 log difference between runs). While the risk of TSE contamination of HSA is only theoretical, these data provide a high degree of assurance that in the event of an adventitious TSE contamination of the HSA used in the ReFacto cell culture process, the purification steps would remove the infectious agent to safe levels.