High-level expression and preparation of recombinant human fibrinogen as biopharmaceuticals.

Fibrinogen is a large and complex glycoprotein containing two sets of each of three different chains (α, ß 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, ß 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.

High-yield preparation of recombinant human α-thrombin for therapeutic use.

In this study, we established stable cell lines producing 1.5 mg/mL recombinant human prothrombin in 400-L fed-batch culture, using CHO DG44 cells as a host cell line. And we also established a recombinant human α-thrombin purification process that produces a purified product suitable for use as a biopharmaceutical, by using recombinant ecarin from CHO DG44 cells, achieving a total yield of approximately 27% of prothrombin in culture medium. The establishment of stable cell lines with high expression levels, long-term passage stability and satisfactory scale-up are essential to ensure the stable supply of biopharmaceuticals. Furthermore, biopharmaceuticals must be of high quality to assure safety and effectiveness in target applications. We had previously reported that recombinant human prethrombin-2 expression level in a stable cell line established using the mouse myeloma cells, Sp2/0-Ag14, reached 200 µg/mL using animal-free materials in 50-L fed-batch culture. However, the productivity was insufficient to completely replace α-thrombin in human plasma preparations. By employing CHO DG44 cells as a host cell line, we had established a stable cell line and achieved significant improvements in quality, productivity of recombinant human α-thrombin manufacture suitable for use as a biopharmaceutical.

High-yield production and characterization of biologically active recombinant aprotinin expressed in Saccharomyces cerevisiae.

Aprotinin is a polypeptide composed of 58 amino acid residues and has a molecular weight of 6512Da. The 58 amino acid residues are arranged in a single polypeptide chain, which is cross-linked by three disulfide bridges and folded to form a pear-shaped molecule. To express recombinant aprotinin in Saccharomyces cerevisiae, a synthetic gene encoding aprotinin was constructed and fused in frame with the pre-sequence of the S. cerevisiae MATalpha1 gene at the cleavage site of signal peptidase. The expression of aprotinin in S. cerevisiae was carried out using the PRB1 promoter. Aprotinin was secreted as a biologically active protein at a concentration of 426 mg/L into high cell density fermentation medium of 70.9 g/L cell dry weight. The purification process consisted of only three major steps and provided consistent yields of recombinant aprotinin using gel filtration high-pressure liquid chromatographic (HPLC) with a purity level higher than 99% and was free of non-aprotinin-related impurities. The recombinant aprotinin had the same characteristics as bovine aprotinin in a number of analytical methods, including alpha2-plasmin inhibition assay, amino acid composition, N-terminal amino acid sequence determination, and mass spectrum analysis. With further optimization of the purification process and culture conditions for high-yield production by S. cerevisiae, this source of recombinant aprotinin may be a promising approach for the commercial manufacture of aprotinin for pharmaceutical use instead of bovine aprotinin.