Transmissible spongiform encephalopathy agent clearance by the immunoaffinity and anion-exchange chromatography steps of the ReFacto manufacturing process.
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.
Booth
J
J
Purification Process Development, Wyeth Biotech, 1 Burtt Road, Andover, MA 01810, USA. jbooth@wyeth.com
Vicik
S
S
Tannatt
M
M
Gallo
C
C
Kelley
B
B
D000818:Animals; D000838:Anions; D001921:Brain / Q000821:virology; D002846:Chromatography, Affinity; D006224:Cricetinae; D005169:Factor VIII / Q000737:chemistry*; D006801:Humans; D023421:Models, Animal; D017096:Prion Diseases / Q000517:prevention & control* / Q000635:transmission; D011328:Prions
D000838:Anions; D011328:Prions; C427184:recombinant factor VIII SQ; D005169:Factor VIII