Biochemical characterization and immunogenicity of Neureight, a recombinant full-length factor VIII produced by fed-batch process in disposable bioreactors

Abstract

Hemophilia A is a X-linked recessive bleeding disorder consecutive to the lack of circulating pro-coagulant factor VIII (FVIII). The most efficient strategy to treat or prevent bleeding in patients with hemophilia A relies on replacement therapy using exogenous FVIII. Commercially available recombinant FVIII are produced using an expensive perfusion technology in stainless steel fermenters. A fed-batch fermentation technology was recently developed to produce 'Neureight', a full-length recombinant human FVIII, in Chinese hamster ovary (CHO) cells. Here, we investigated the structural and functional integrity and lack of increased immunogenicity of Neureight, as compared to two commercially available full-length FVIII products, Helixate and Advate, produced in baby hamster kidney or CHO cells, respectively. Our results demonstrate the purity, stability and functional integrity of Neureight with a standard specific activity of 4235 ± 556 IU/mg. The glycosylation and sulfation profiles of Neureight were similar to that of Advate, with the absence of the antigenic carbohydrate epitopes α-Gal and Neu5Gc, and with sulfation of Y1680, that is critical for FVIII binding to von Willebrand factor (VWF). The endocytosis of Neureight by human immature dendritic cells was inhibited by VWF, and its half-life in FVIII-deficient mice was similar to that of Advate, confirming unaltered binding to VWF. In vitro and in vivo assays indicated a similar immunogenicity for Neureight, Advate and Helixate. In conclusion, the production of full-length FVIII in a fed-batch fermentation mode generates a product that presents similar biochemical, functional and immunogenic properties as products developed using the classical perfusion technology.

Perfusion process combining low temperature and valeric acid for enhanced recombinant factor VIII production

“…Additionally, FVIII undergoes a very large number of post-translational modifications. 17 For improving recombinant FVIII production, a screening for cultivation conditions in batch was done by our group and significant increases in q p were obtained at low temperatures with short-chain fatty acids supplementation. 14 Since the commercialization of the first recombinant FVIII in 1992, a wide range of products has been marketed.…”

“…15 Due to FVIII instability, industrial processes for rFVIII production are operated in perfusion mode to allow for short residence time in the bioreactor, 16 although a new FVIII product that is under development has been recently reported to be produced in fed-batch mode. 17 For improving recombinant FVIII production, a screening for cultivation conditions in batch was done by our group and significant increases in q p were obtained at low temperatures with short-chain fatty acids supplementation. 18 Although both valeric acid and butyric acid led to increased q p , the use of valeric acid had the advantage of being less cytotoxic.…”

Identification of Key Coagulation Activity Determining Elements in Canine Factor VIII

“…infusion of recombinant or plasma-derived FVIII protein. 4,5 While this replacement treatment corrects the abnormal bleeding phenotype, it is life-long and timeconsuming 6 and is estimated to cost from $150,000 to $300,000 per patient per year in the United States. 7 Therefore, the development of a FVIII protein with increased activity would be valuable and could potentially enhance the quality of life for HA patients.…”