Galectin 3-binding protein is a potential contaminant of recombinantly produced factor IX.

Haemophilia B, or factor IX (FIX) deficiency, represents 15% of the hereditary haemophilias. The serious morbidity from the transmission of infectious agents in plasma-derived material has mandated a need for the production of recombinant product. The rate-limiting step for the production of recombinant FIX is gamma-carboxylation, a post-translational modification carried out only in mammalian cells. To test the carboxylation efficiency of recombinantly produced FIX in vitro and to improve the isolation of the pure active product, we produced FIX in a transfected human cell line (293 human embryonic kidney cells) and isolated material by immunoaffinity chromatography followed by hydroxyapatite chromatography. Unexpectedly, during hydroxyapatite chromatography, we discovered that purified FIX was contaminated by a heretofore unknown protein. Further analysis by mass spectrometry (MS) sequencing revealed this protein to be galectin-3-binding protein (G3BP). The above results raise an important note of caution regarding the production of recombinant FIX and, indeed, other proteins produced recombinantly in mammalian cells.