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.

Keywords: CHO DG44; biopharmaceuticals; high-level expression; high-recovery preparation; recombinant human fibrinogen.

Fig. 1

Map of the expression plasmid SPNFH-ArsFF-Fbg αγβγ for human fibrinogen. The CAGG expression cassettes containing human fibrinogen α, β and γ genes enclosed by Ars insulators were arranged in the order of α, γ, β and γ cassettes.

Fig. 2

Cell proliferation and fibrinogen expression. (A) Cell density (triangles) and viability (circles) were monitored for 16 days. (B) Expression levels of recombinant fibrinogen were quantified by ELISA (diamond symbols). (C) Recombinant fibrinogens in culture supernatants of Days 7–15 were estimated using 10% SDS-PAGE under reducing conditions and CBB staining. Fibrinogen α chain (α), fibrinogen β chain (β) and fibrinogen γ chain (γ) are shown by arrow heads.

Fig. 3

SDS-PAGE and western blotting (WB) of recombinant fibrinogen. (A) Under reducing and non-reducing conditions, recombinant fibrinogen and plasma fibrinogen were estimated by 5–20% gradient SDS-PAGE stained with CBB. (B) Under reducing conditions, α, β and γ chains are shown by WB using fibrinogen chain-specific antibodies. pFbg, plasma fibrinogen; rFbg, recombinant fibrinogen.

Fig. 4

Carbohydrate analysis of recombinant and plasma fibrinogens. (A) Molecular weight changes of fibrinogens treated with peptide-N-glycosidase F (PNGase F) were evaluated by 5–20% gradient SDS-PAGE stained with CBB and WB using fibrinogen chain-specific antibodies. Samples 1 and 2 are plasma fibrinogen treated without and with PNGase F, respectively. Samples 3 and 4 are recombinant fibrinogen treated without and with PNGase F, respectively. (B) Chains of reduced fibrinogen were separated and analysed by LC/MS. Data of deconvoluted mass spectra for each of the fibrinogen chains are shown, and differences in the molecular weight following PNGase F treatment are indicated in parentheses. pFbg, plasma fibrinogen; rFbg, recombinant fibrinogen.

Fig. 5

Polymerization evaluation of recombinant fibrinogen by monitoring changes in turbidity. Polymerization was initiated by addition of thrombin (0.05 U/ml) at time 0 under FXIII-containing conditions (0.5 U/ml) to 2 mg/ml of recombinant fibrinogen (broken line) or plasma-derived FXIII-depleted fibrinogen (solid line), and polymerization was evaluated by monitoring changes in turbidity at 450 nm for 30 min (N = 2).

Fig. 6

SEM images of fibrin fibres. Fibrin clots were formed under FXIII-containing conditions (0.5 U/ml) by addition of thrombin (0.05 U/ml) to recombinant or plasma-derived FXIII-depleted fibrinogen (2 mg/ml). Clots were fixed, dehydrated, lyophilized, coated with osmium and imaged by SEM. The magnification bar represents 1 μm. pFbg, plasma fibrinogen; rFbg, recombinant fibrinogen.

Fig. 7

Release of fibrinopeptides A and B from recombinant fibrinogen and plasma fibrinogen. The release of fibrinopeptide A is shown in panel A and that of fibrinopeptide B is shown in panel B. Each of the fibrinopeptides was quantitated by HPLC using absorbance at 210 nm (N = 2). Circles represent plasma fibrinogen, and diamonds represent recombinant fibrinogen.

Fig. 8

Changes in the form of fibrinogen chains in cross-linking reactions and viscoelastic evaluation of fibrin clots. At time 0, thrombin (1 U/ml) was added to a mixture of FXIII (1 U/ml) and plasma or recombinant fibrinogen (2 mg/ml). The reactions were stopped at set times (0, 5, 10 and 30 min) by addition of SDS-PAGE buffer containing 4 M urea and boiling. Fibrinogen Samples 1–8 were electrophoresed on 10% SDS-PAGE under reducing conditions and stained with CBB (A), followed by WB using anti-α chain (B), anti-β chain (C; anti-fibrinopeptide B) and anti-γ chain (D) antibodies. Lanes 1–4 are plasma fibrinogen and 5–6 are recombinant fibrinogen. Lanes 1 and 5 are 0 time samples. Lanes 2 and 6 are samples after 5 min. Lanes 3 and 7 are samples after 10 min. Lanes 4 and 8 are samples after 30 min. (E) Fibrin clots were reacted for 30 min without stopping the reaction, and the elastic modulus was measured using an EZ-Test EZ-S table-top universal tester (N = 5). pFbg, plasma fibrinogen; rFbg, recombinant fibrinogen.