Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics.

N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient's bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4 and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties.

Comparative N-Glycosylation Analysis of the Fc Portions of a Chimeric Human Coagulation Factor VIII and Immunoglobulin G1.

Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion to Fc-immunoglobulin (Ig)G that mediate protection from clearance or degradation via binding to the neonatal Fc receptor. While human-like N-glycosylation of recombinant FVIII is known to be crucial for the clotting factor's quality and function, the particular glycosylation of the fused Fc portion has not been investigated in detail so far, despite its known impact on Fcγ receptor binding. Here, we analyzed the N-glycosylation of the Fc part of a chimeric FVIII-Fc protein compared to a commercial IgG1 purified from human plasma. Fc parts from both samples were released by enzymatic cleavage and were subsequently separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Corresponding protein bands were referred to PNGase F in-gel digestion in order to release the respective N-glycans. Analysis via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed structural differences of both N-glycan patterns. Labeling with 2-aminobenzamide (2AB) and analysis via hydrophilic interaction liquid chromatography (HILIC) allowed a quantitative comparison of the respective N-glycosylation. Observed variations in Fc glycosylation of the chimeric FVIII fusion protein and human plasma-derived IgG1, e.g., regarding terminal sialylation, are discussed, focusing on the impact of the clotting factor's properties, most notably its binding to Fcγ receptors.

ADAMTS13 content and VWF multimer and triplet structure in commercially available VWF/FVIII concentrates.

ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) into smaller multimers in vivo. This cleavage creates both the typical multimeric size distribution and the characteristic triplet band distribution of VWF. Here we analysed ADAMTS13 content, VWF multimeric size distribution and VWF triplet structure in five commercial VWF/factor VIII (FVIII) concentrates. The relative distribution of ADAMTS13 activity values corresponded well to the ADAMTS13 antigen values for all examined concentrates except Haemate HS®, which had markedly higher ADAMTS13 antigen/activity ratio, with Fanhdi® and Haemate HS® displaying the most intense ADAMTS13 signal. Interestingly, ADAMTS13 levels did not correlate with the high molecular weight multimer content of the concentrates, but did correlate with VWF triplet distribution. Densitometric quantification showed that Wilate®, Immunate® and Willfact® displayed human plasma-like VWF triplet distribution, whereas Fanhdi® and Haemate HS® showed enhanced content of the faster migrating triplet band, which corresponded well to their higher ADAMTS13 content. In summary, Immunate®, Willfact® and Wilate® had lower levels of ADAMTS13 antigen and activity and exhibited a plasma-like VWF triplet structure. Fanhdi® and Haemate HS® had higher ADAMTS13 content and an altered triplet structure. The possible impact of these observations on function and clinical efficacy of VWF/FVIII concentrates is discussed.

Distinct role of von Willebrand factor triplet bands in glycoprotein Ib-dependent platelet adhesion and thrombus formation under flow.

Multimeric glycoprotein von Willebrand factor (VWF) exhibits a unique triplet structure of individual oligomers, resulting from ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13) cleavage. The faster and slower migrating triplet bands of a given VWF multimer have one shorter or longer N-terminal peptide sequence, respectively. Within this peptide sequence, the A1 domain regulates interaction of VWF with platelet glycoprotein (GP)Ib. Therefore, platelet-adhesive properties of two VWF preparations with similar multimeric distribution but different triplet composition were investigated for differential functional activities. Preparation A was enriched in intermediate triplet bands, whereas preparation B predominantly contained larger triplet bands. Binding studies revealed that preparation A displayed a reduced affinity for recombinant GPIb but an unchanged affinity for collagen type III when compared to preparation B. Under high-shear flow conditions, preparation A was less active in recruiting platelets to collagen type III. Furthermore, when added to blood from patients with von Willebrand disease (VWD), defective thrombus formation was less restored. Thus, VWF forms lacking larger-size triplet bands appear to have a decreased potential to recruit platelets to collagen-bound VWF under arterial flow conditions. By implication, changes in triplet band distribution observed in patients with VWD may result in altered platelet adhesion at high-shear flow.