Recombinant AnFIX expression and characterization. Recombinant AnFIX expression was determined in transient (A-B) and stable (C) mammalian cell expression systems with and without R338L (Padua) mutation (B-C) by measuring FIX activity in the conditioned medium by 1-stage coagulation assay and manually counting the cells present in each well by hemocytometer to obtain a FIX activity (IU) accumulated per million cells per 24 hours (expression value). Error bars indicate sample standard deviation. Closed shapes represent individual clones, with sample size in parentheses and mean as a dashed line. Statistical analysis was performed using a 1-way analysis of variance with post hoc Tukey multiple comparisons test. (D) Highly purified recombinant hFIX, hFIX-Padua, An96, and An96-Padua were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and visualized by Coomassie blue staining under reducing (+) and nonreducing (−) conditions after incubation at 95°C for 5 minutes with or without dithiothreitol, respectively. *AnFIX variants with significant expression above the other variants (P < .05).