We transfected each of the PSG1-encoding plasmids described above into dihydrofolate reductase-negative (DHFR À ) CHO cells along with pDCHIP, which encodes the Dhfr minigene, in a 1:10 molar ratio using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Positive transformants were obtained by methotrexate selection until maximum levels of protein were obtained from the cells, as determined by ELISA of the collected supernatants. Stably transfected cells were seeded into 5-kDa molecular weight cutoff hollow-fiber cartridges (FiberCell Systems, Inc.) and grown in Dulbecco modified Eagle medium supplemented with 2% fetal bovine serum (FBS; low IgG for PSG1-Fc production), 10 mM Hepes, 50 IU/ml of penicillin, and 50 lg/ ml of streptomycin. The supernatants from the cartridges were harvested daily, centrifuged at 5000 3 g for 10 min to remove cell debris, and then kept frozen until processed as described below. The control protein, FLAG-Fc, was harvested from the supernatant of DHFR À CHO cells that were a kind gift from Dr. Gerardo Kaplan (Center of Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD).