Mutations abolishing or substantially reducing coagulation factor IX (FIX) function lead to hemophilia B, a hereditary bleeding disorder that can be managed by replacement therapy using plasma-derived or recombinant FIX. For prophylaxis, 2-weekly infusions are traditionally recommended. Thus, a modified FIX protein with prolonged circulatory half-life allowing less frequent infusions may improve patient convenience and compliance. Several strategies for reducing clearance of FIX have been described. Recombinant FIX compounds prolonged by glycoPEGylation [1], genetic fusion to albumin [2] or genetic fusion to the IgG Fc domain [3] are currently in clinical development.In the present study, we investigated the possibility of prolonging FIX by introducing new N-glycans. Previous studies suggest a possible role for N-glycans in determining the clearance of FIX. Enzymatic removal of N-glycans increased clearance of wild-type recombinant FIX [4,5], and a correlation between N-glycan sialic acid content and terminal half-life of recombinant FIX has been reported [6]. N-glycosylation is anintracellular processcarriedoutby the oligosaccharyl transferase complex, which glycosylates asparagine residues in N-X-S/T motifs of nascent proteins. Thus, N-glycans can be added to a recombinant protein by introducing mutations that establish N-glycosylation motifs in the amino acid chain. This strategy has previously proven efficient in prolonging the circulatory half-lives of erythropoietin, follicle stimulating hormone, interferon alpha and growth hormone [7].Wild-type FIX has two N-glycans at positions 157 and 167. We introduced additional N-glycosylation sites in the FIX protein by site-directed mutagenesis of human FIX-coding cDNA. The extra N-glycosylation sites were introduced at positions that we believed allowed for the presence of a glycan without severely interfering with the biological functions of FIX. An expression vector encoding FIX-T172N-K228N-I251T-A262T was established. Besides the wild-type glycosylation sites, the FIX-T172N-K228N-I251T-A262T variant also has N-glycosylation sites at amino acid positions 172, 228, 249 and 260 and thus contains a total of six N-glycosylation sites. Clonal CHO-K1 cell lines producing FIX-T172N-K228N-I251T-A262T were generated using the procedures previously described for FVII variants [8]. A clone exhibiting a favourable combination of FIX productivity and degree of Gla domain gamma-carboxylation was selected, and FIX-T172N-K228N-I251T-A262T was purified from cell culture supernatant as previously described for wild-type FIX [1]. This purification procedure combines an immunoaffinity step using a monoclonal antibody recognizing the functional conformation of the FIX Gla domain and an anion exchange chromatography step, allowing selective isolation of fractions with a desired gammacarboxylation profile. Only FIX with 10-12 gamma-carboxy groups have fully functional Gla domains [9], and according to anion exchange HPLC as previously described [1], the Gla domains of the FIX-T172N-K2...