Purification and characterization of a new recombinant factor VIII (N8)

Abstract

A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg(-1). Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.

Factor VIII C1 Domain Spikes 2092–2093 and 2158–2159 Comprise Regions That Modulate Cofactor Function and Cellular Uptake

“…For these studies, recombinant FVIII Turoctocog alfa (previously named N8) was produced in CHO cells as described previously (31). The molecule consists of a heavy chain of 88 kDa including a 21-amino acid residue-truncated B domain and a light chain of 79 kDa.…”

“…The molecule consists of a heavy chain of 88 kDa including a 21-amino acid residue-truncated B domain and a light chain of 79 kDa. It contains four N-glycosylation sites of which two are complex biantennary glycans and two are high-mannose structures (31). FVIII and KM33 were dialyzed to a buffer comprising 20 mM imidazole (pH 7.3), 10 mM CaCl 2 , and 150 mM NaCl, and the proteins were kept at 2°C until the start of the experiment.…”

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues

“…Sequence reactions were performed with BigDye terminator sequencing kit (Applied Biosystems, Foster City, CA). Expression of Recombinant Proteins-Recombinant factor VIII (FVIII) Turoctocog alfa (previously named N8) was produced in CHO cells as described previously (33). The molecule consists of a heavy chain of 88 kDa including a 21-amino acid residue truncated B-domain and a light chain of 79 kDa; it contains four N-glycosylation sites of which two are complex biantennary glycans and two are high-mannose structures.…”

A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models

“…13 Arthrobacter ureafaciens sialidase was produced as a His 6 -tagged truncated form in a BL21-derived E. coli strain. 17 Tick anticoagulant protein 18,19 and turoctocog alfa (previously named N8) 14 were produced as described. Advate was from Baxter, Westlake Village, CA, and ReFacto from Wyeth, Philadelphia, PA.…”

“…The specific activity was calculated by dividing the activity of the samples with the protein concentration determined by high-performance liquid chromatography as described. 14,24 Binding to VWF and lipoprotein-receptor-related protein (LRP)…”