Hyperglycosylation prolongs the circulation of coagulation factor IX

Abstract

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...

Recent developments in protein and peptide parenteral delivery approaches

“…Bolt et al showed that hyperglycosylation of recombinant Factor IX, a blood coagulation factor used to treat Hemophilia B, improved and prolonged its systemic circulation time relative to native protein [43]. Pharmacokinetic profile of recombinant human IFN-α2 (rhIFN-α2) was also remarkably improved by N -glycosylation.…”

The Relationship of Secretion and Activity of Recombinant Factor IX with N-Glycosylation

“…Although the effect of FIX N-glycans on protein performance is unclear yet, few studies have focused on the N-glycans effect on FIX function. A possible role for N-glycans in determining the clearance of FIX suggests that hyper-glycosylation decreases clearance and prolongs the circulation of FIX [19]. Moreover, although the enzymatic removal of sialic acid was reported to increase the clearance of wild-type recombinant FIX, no effect was observed on its pre-coagulation activity [26].…”

“…Since both N-glycosylation sites occur in the activation domain and are absent in the active enzyme, the enzymatic activity of FIX may not be affected by the N-glycan structures. However, other essential functions have not been considered suggesting that the N-glycans are necessary for the prolongation of circulating zymogen [17][18][19]. On the other hand, N-linked glycosylation is principally vital because of influencing some properties such as folding and secretion [20].…”

Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

“…3). While g-carboxylation is a key PTM on FIX, other PTMs also have important functional roles and their composition and abundance can also be dependent on changes in the expression system and bioprocess parameters [3,5,9,10,14,21,27,68,92,[94][95][96][97][98][99]. Therefore, it would be advantageous to be able to predict the relative abundance and composition of different types of PTMs on purified rFIX by analyzing the culture supernatant.…”

“…,27,68,92,[94][95][96][97][98][99]. For example, rFIX N-linked glycans (native and engineered) influence rFIX in vivo half-life[94,[116][117][118][119] and may modulate rFIX activation[31], while rFIX EGF-like 1 Oglycans stabilize the domain and likely participate in protein-protein interactions[92,99]. Changes in the expression system and incubation conditions alter the number and variety of PTMs in recombinant proteins[7,10,11,21,70,109,114], and new, unexpected PTMs may appear.…”