Characterization of recoded FIX constructs expressed from lentiviral transduction stable expression system. (A) The mRNA and antigen expression levels of wild-type (WT), OPT-1A, and OPT-2A variant lentiviral-transduced stable expression clones selected for further characterization. Data are represented as mean ±  standard deviation (SD). (B) CD analysis of FIX secondary structures. Recoded FIX constructs showed similar CD spectra profiles that are distinct from WT FIX. (C) Limited trypsin digestion profiles of WT, OPT-1A, and OPT-2A constructs. Both OPT-1A and OPT-2A constructs showed increased sensitivity to trypsin digestion compared with WT FIX (highlighted by red color squares). (D-E) Inhibitory antibody assay data from assays employing FIX-depleted plasma spiked with polyclonal anti-FIX antibodies and hemophilia B patient with anti-FIX inhibitory antibodies, respectively. Data showed significantly different inhibition kinetics for OPT-1A and OPT-2A FIX in comparison with wild type when incubated with the spiked plasma (panel D, with ED₅₀ 0.0284, 0.0339, and 0.0286 for WT, OPT-1A, and OPT-2A) and hemophilia B patient plasma (panel E, with ED₅₀ 0.0637, 0.0580, and 0.0453 for WT, OPT-1A, and OPT-2A), respectively. Together, data in panels B-E demonstrated conformational differences of recoded FIX constructs relative to WT FIX. (F) Specific activity assessment data for recoded FIX constructs presented as fold activity over WT. Data are represented as mean ± SD. (G) Propeptide processing and γ-carboxylation profiles of purified FIX from selected clones.