An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of f1 AM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3 -4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials. B 2004 Wiley Periodicals, Inc.

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West Nile Virus (WNV) was immediately and completely inactivated to below assay detection limits upon addition of solvent/detergent (S/D) to intermediate process pools of second and third generation B-domain deleted recombinant Factor VIII (BDDrFVIII; ReFacto, Wyeth, Cambridge, MA, USA). We verify that the S/D step provides robust enveloped virus inactivation (>5 log(10)) and functions as a WNV barrier.

ReFacto (moroctocog alfa), a recombinant factor VIII approved for the treatment of haemophilia A, is produced by a mammalian cell-culture process that includes therapeutic-grade human serum albumin (HSA) in the cell-culture medium. While to date there have been no cases of transmissible spongiform encephalopathy (TSE) resulting from the clinical use of HSA, Wyeth conducted a study to demonstrate that the ReFacto manufacturing process has significant capacity to remove a TSE agent if it were present as a contaminant in the HSA. The immunoaffinity (8A4 Sepharose) and anion-exchange (Q Sepharose) chromatography steps were evaluated for the clearance of the hamster TSE agent, strain 263K. This Good Laboratory Practice study was performed using appropriately qualified, laboratory-scale chromatography systems. Filtered brain homogenate from TSE-infected hamsters was added to loads of both chromatographic columns, and the concentration of TSE agent in the loads and product pools were determined using a validated western blot quantitation method. Replicate chromatography runs were consistent, as demonstrated by the < or =0.7 log(10) difference observed in TSE agent reduction between each pair of runs. The immunoaffinity and anion-exchanges steps demonstrated 3.8 log reduction and >5.2 log reduction respectively. These data provide a high degree of assurance that in the unlikely event of a TSE contamination of the HSA used in the ReFacto cell-culture process, the purification steps have the potential to remove the infectious agent to extremely low levels, thereby significantly reducing the risk to patients receiving ReFacto.

No 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.