title: High-level expression and preparation of recombinant human fibrinogen as biopharmaceuticals.
abstract: Fibrinogen is a large and complex glycoprotein containing two sets of each of three different chains (α, β and γ). There have been no reports of high-level expression of fibrinogen at commercial levels using mammalian cultured cells such as CHO cells because of the difficulty in highly expressing a protein with such a complex structure. We achieved high-level (1.3 g/l or higher) expression of recombinant human fibrinogen using CHO DG44 cells by optimizing the expression system and culture conditions. We also succeeded in establishing a high-recovery preparation method for recombinant fibrinogen that rarely yields degraded products. To characterize the properties of the recombinant human fibrinogen, we performed SDS-PAGE; western blotting of the α, β and γ chains using specific antibodies and scanning electron microscopy observations of fibrin fibres. We also evaluated the functional equivalence between recombinant fibrinogen and plasma fibrinogen with respect to the release of fibrinopeptides initiated by thrombin and its cross-linking properties. The basic properties of recombinant fibrinogen showed no apparent differences from those of plasma fibrinogen. Here, we report the development of methods for the culture and preparation of recombinant human fibrinogen of satisfactory quality that can be scaled up to the commercial level.
authors.affiliation: R&D Division; and.
authors.forename: Masaki
authors.initials: M
authors.lastname: Hirashima
authors.affiliation: R&D Division; and.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Imamura
authors.affiliation: Blood Plasma Division, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1314-1 Kyokushi-Kawabe, Kikuchi, Kumamoto 869-1298, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Yano
authors.affiliation: R&D Division; and.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Kawamura
authors.affiliation: R&D Division; and.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Meta
authors.affiliation: Blood Plasma Division, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1314-1 Kyokushi-Kawabe, Kikuchi, Kumamoto 869-1298, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Tokieda
authors.affiliation: R&D Division; and nakashima-to@kaketsuken.or.jp.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Nakashima
mesh_terms: D000818:Animals; D001688:Biological Products / Q000302:isolation & purification* / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000096:biosynthesis* / Q000302:isolation & purification*; D006801:Humans; D008855:Microscopy, Electron, Scanning; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*; D013917:Thrombin / Q000378:metabolism
chemical_list: D001688:Biological Products; D011994:Recombinant Proteins; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin
keywords: CHO DG44; biopharmaceuticals; high-level expression; high-recovery preparation; recombinant human fibrinogen