Characterization of recoded FIX constructs expressed from Flp-In system. (A) The mRNA and antigen expression levels of wild-type (WT), OPT-1A, and OPT-2A variants from single copy expression Flp-in system stable expression cell lines. Data are represented as mean ± standard deviation (SD). (B) Propeptide processing and γ-carboxylation profiles of purified FIX. (C) Specific activity data for recoded FIX constructs presented as fold activity over WT. Data are represented as mean ± SD. Data in panels B and C demonstrated relatively higher propeptide FIX fraction, lower Gla, and lower specific activity for FIX expressed from the variant with the highest antigen expression, OPT-1A. This data suggested suboptimal posttranslational processing of FIX when overexpressed.