Broadband complementary vibrational spectroscopy with cascaded intra-pulse difference frequency generation.

One of the essential goals of molecular spectroscopy is to measure all fundamental molecular vibrations simultaneously. To this end, one needs to measure broadband infrared (IR) absorption and Raman scattering spectra, which provide complementary vibrational information. A recently demonstrated technique called complementary vibrational spectroscopy (CVS) enables simultaneous measurements of IR and Raman spectra with a single device based on a single laser source. However, the spectral coverage was limited to ∼1000cm-1, which partially covers the spectral regions of the fundamental vibrations. In this work, we demonstrate a simple method to expand the spectral bandwidth of the CVS with a cascaded intra-pulse difference-frequency generation (IDFG). Using the system, we measure broadband CVS spectra of organic liquids spanning over 2000cm-1, more than double the previous study.

Potent Hemostatic Efficacy of a Novel Recombinant Fibrin Sealant Patch (KTF-374) in Rabbit Bleeding Models.

PURPOSE: Fibrin sealants are used for hemostasis during surgery. Commercially available fibrin sealants are made of materials of human or animal origin. We developed a novel recombinant fibrin sealant patch (KTF-374) that has thin and flexible properties. This study evaluated the hemostatic efficacy of KTF-374 for various patterns of bleeding in rabbits, as compared with that of the existing fibrin-coated collagen fleece (FCCF). MATERIALS AND METHODS: Test hemostats used were KTF-374 and FCCF. Laparotomy was performed under general anesthesia in rabbits. We created wounds in the liver, caudal vena cava, and ventral aorta under anticoagulating conditions with heparin. Test hemostats were then applied to the wound site and compressed manually for 3 min. Hemostatic efficacy was evaluated with the success rate of hemostasis at 3 min. RESULTS: In all bleeding models, the success rate of hemostasis was significantly higher with KTF-374 than FCCF. The hemostatic success rate of KTF-374 and FCCF was 100% vs. 25% (p = .007) in the partial hepatectomy model (n = 8); 100% vs. 12.5% (p = .001) in the caudal vena cava resection model (n = 8); and 100% vs. 25% (p = .004) in the ventral aortic puncture model (n = 8). The wound site could clearly be recognized through the patch after the application of KTF-374 but not FCCF. CONCLUSIONS: These results suggest that KTF-374 possesses more potent hemostatic properties than FCCF for various patterns of bleeding. KTF-374 is a promising hemostat due to its potent efficacy and good visibility of the wound site through the patch.

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.

Crystal structure of thrombin in complex with S-variegin: insights of a novel mechanism of inhibition and design of tunable thrombin inhibitors.

The inhibition of thrombin is one of the important treatments of pathological blood clot formation. Variegin, isolated from the tropical bont tick, is a novel molecule exhibiting a unique 'two-modes' inhibitory property on thrombin active site (competitive before cleavage, noncompetitive after cleavage). For the better understanding of its function, we have determined the crystal structure of the human α-thrombin:synthetic-variegin complex at 2.4 Å resolution. The structure reveals a new mechanism of thrombin inhibition by disrupting the charge relay system. Based on the structure, we have designed 17 variegin variants, differing in potency, kinetics and mechanism of inhibition. The most active variant is about 70 times more potent than the FDA-approved peptidic thrombin inhibitor, hirulog-1/bivalirudin. In vivo antithrombotic effects of the variegin variants correlate well with their in vitro affinities for thrombin. Our results encourage that variegin and the variants show strong potential for the development of tunable anticoagulants.

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.

Hemostatic efficacy of a recombinant thrombin-coated polyglycolic acid sheet coupled with liquid fibrinogen, evaluated in a canine model of pulmonary arterial hemorrhage.

BACKGROUND: In thoracic surgery, although infrequent, we encounter unexpected damage to the pulmonary artery (PA). In the present study, we evaluated the hemostatic efficacy of a newly developed fibrin-based sheet material, thrombin sheet, coupled with liquid fibrinogen (TSF), in an experimental model of PA hemorrhage. METHODS: Female beagles (n = 8) were used for the study. Left thoracotomy was performed under general anesthesia. PA injury (approximately 4 x 2 mm) was created, and repaired by TSF (TSF group) or TachoComb (TC group). The animals were allowed to survive, and the repaired site was evaluated 4 weeks after the experiment. RESULTS: The number of sheet application and compression procedures required for hemostasis was increased in the TC group compared with in the TSF group (TC vs. TSF, 4 +/- 1 vs. 1 +/- 0.5, p = 0.01, unpaired t test). The time required to achieve hemostasis was increased in the TC group compared with in the TSF group (TC vs. TSF, 7 +/- 3 vs. 1 +/- 0.5 minutes, p = 0.01, unpaired t test). The amount of bleeding during the hemostasis procedure was increased in the TC group compared with in the TSF group (TC vs. TSF, 48 +/- 22 vs. 3 +/- 3 g, p = 0.01, unpaired t test). At 4 weeks, rethoracotomy revealed no apparent indication of delayed bleeding, such as intrathoracic hematoma formation or excessive adhesion formation in the vicinity of PA, in either group. Histologically, the vessel lumen was well sustained in both groups, with no apparent stenosis or thrombus formation. CONCLUSION: The hemostatic efficacy of TSF was superior to TC in this particular experiment. Single application of TSF was sufficient to achieve hemostasis in all but one animal. Compression time of approximately 1 minute was also very short albeit that the bleeding was from the PA and not an artery. These results were presumably because the adhesion was stronger, faster, and the sheet was more pliable in TSF compared with TC.

Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin.

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.