Abstract
Background & MethodsRecombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications.Results & DiscussionThe analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures.ConclusionsFrom all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0205-1) contains supplementary material, which is available to authorized users.
Highlights
Background & MethodsRecombinant factor VII, the precursor molecule for recombinant activated Coagulation factor VII (FVII), is, due to its need for complex post translational modifications, produced in mammalian cells
From all Recombinant factor VII (rFVII) isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal N-acetyl galactosamine (GalNAc), and Chinese hamster ovary (CHO) cells were assumed to be the best option for the production of rFVII
Expression of rFVII in mammalian and human cell lines Human rFVII derived from baby hamster kidney (BHK), CHO, and HEK293 cells was produced at laboratory scale, and purified from culture supernatants
Summary
Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and with those of plasma derived FVII (pdFVII), using various analytical methods. FVII carries complex post-translational protein modifications (PTMs) including γ-carboxylation, N- and O-. The modifications of rFVIIa produced in Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells have been described to differ from those of pdFVII or FVIIa, especially in N-glycosylation.
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