Galectin-3 Binding Protein Contaminates the Purification of Recombinant Factor IX.

The hemophilas are the most common severe hereditary bleeding disorders. The standard treatment of these disorders is to infuse plasma concentrates of the missing coagulation factor. However, the high cost of replacement therapy and the serious morbidity from the transmission of infectious agents have mandated a need for alternative therapeutic strategies. One of these strategies is gene therapy and we embarked on a novel gene therapeutic strategy involving the use of factor IX-transduced bone marrow stroma to treat hemopihlia B, a deficiency of the gamma-carboxylated protein, factor IX. The rate limiting step for the production of factor IX is gamma-carboxylation, a post translational modification carried out only in mammalian cells. In order to test the carboxylation efficiency of our gene therapeutic platform, we first transfected Human Embryonic Kidney (HEK) 293 cells with a retroviral construct containing factor IX. Factor IX was purified by immunoaffinity chromatography and then subject to hydroxyapatite chromatography to isolate biologically active properly carboxylated factor IX from inactive uncarboxylated factor IX. Unexpectedly, during hydroxyapatite chromatography, we discovered that purified factor IX was contaminated by a heretofore unknown protein. Further analysis by mass spectrometry sequencing revealed this protein to be galectin-3 binding protein (G3BP).G3BP contaminates media of HEK293 cells regardless of whether factor IX is transfected into these cells. Furthermore, the contamination of purified media is not specific for factor IX as G3BP is isolated regardless of the antibody used for purification. In summary, the above results provide important information regarding the production of recombinant factor IX and uncover a potentially new contaminant in recombinant products produced in mammalian cells.