Prethrombin-1 as a Drug Substance Promoting Hemostasis with Reduced Risk of Thrombosis.

INTRODUCTION: Prethrombin-1 is a Gla-domain lacking enzymatically inactive split product that results from the cleavage of fragment 1 from prothrombin by thrombin in a feedback reaction. METHODS: A prethrombin-1 preparation derived from human plasma was tested for its hemostatic and thrombogenic properties. Animal models of nail clipping (for rabbits) and tail clipping (for mice) were developed to measure blood loss in FVIII-inhibitor or rivaroxaban anticoagulated rabbits and mice, respectively. A modified Wessler test was used in rabbits to assess the thrombogenic potential by Wessler score and clot weight. Studies were performed in groups of three to six for prethrombin-1 dose escalation and comparison with prothrombin, Beriplex®, FEIBA®, and saline as a control. Data were analyzed using t-statistics or the Mann Whitney U test as applicable. RESULTS: Prethrombin-1 has excellent hemostatic properties in anticoagulated mouse and rabbit bleeding models. Wessler tests suggest that in contrast to activated and nonactivated prothrombin complexes, prethrombin-1 has negligible thrombogenic potential. CONCLUSION: The thrombin zymogen prethrombin-1 promotes hemostasis with reduced risk of thrombosis. Prethrombin-1 may have potential to become a life-saving treatment for patients who bleed or are at risk of bleeding.

A microscale method of protein extraction from bacteria: Interaction of Escherichia coli with cationic microparticles.

We developed a simple, highly selective, efficient method for extracting recombinant proteins from Escherichia coli. Our recombinant protein yield was equivalent to those obtained with high pressure homogenization, and did not require exposure to harsh thermal, chemical, or other potentially denaturing factors. We first ground conventional resin, designed for the exchange of small anions, into microparticles about 1µm in size. Then, these cationic microparticles were brought convectively into close contact with bacteria, and cell membranes were rapidly perforated, but solid cell structures were not disrupted. The released soluble components were adsorbed onto the cell wall associated microparticles or diffused directly into the supernatant. Consequently, the selective adsorption and desorption of acidic molecules is built into our extraction method, and replaces the equally effective subsequent capture on anion exchange media. Simultaneously to cell perforation flocculation was induced by the microparticles facilitating separation of cells yet after desorption of proteins with NaCl. Relative to high pressure homogenization, endogenous component release was reduced by up to three orders of magnitude, including DNA, endotoxins, and host cell proteins, particularly outer membrane protein, which indicates the presence of cell debris.

Prediction of inclusion body solubilization from shaken to stirred reactors.

Inclusion bodies (IBs) were solubilized in a µ-scale system using shaking microtiter plates or a stirred tank reactor in a laboratory setting. Characteristic dimensionless numbers for mixing, the Phase number Ph and Reynolds number Re did not correlate with the kinetics and equilibrium of protein solubilization. The solubilization kinetics was independent of the mixing system, stirring or shaking rate, shaking diameter, and energy input. Good agreement was observed between the solubilization kinetics and yield on the µ-scale and laboratory setting. We show that the IB solubilization process is controlled predominantly by pore diffusion. Thus, for the process it is sufficient to keep the IBs homogeneously suspended, and additional power input will not improve the process. The high-throughput system developed on the µ-scale can predict solubilization in stirred reactors up to a factor of 500 and can therefore be used to determine optimal solubilization conditions on laboratory and industrial scale.

Matrix-assisted refolding of autoprotease fusion proteins on an ion exchange column.

Refolding of proteins must be performed under very dilute conditions to overcome the competing aggregation reaction, which has a high reaction order. Refolding on a chromatography column partially prevents formation of the intermediate form prone to aggregation. A chromatographic refolding procedure was developed using an autoprotease fusion protein with the mutant EDDIE from the N(pro) autoprotease of pestivirus. Upon refolding, self-cleavage generates a target peptide with an authentic N-terminus. The refolding process was developed using the basic 1.8-kDa peptide sSNEVi-C fused to the autoprotease EDDIE or the acidic peptide pep6His, applying cation and anion exchange chromatography, respectively. Dissolved inclusion bodies were loaded on cation exchange chromatographic resins (Capto S, POROS HS, Fractogel EMD SO(3)(-), UNOsphere S, SP Sepharose FF, CM Sepharose FF, S Ceramic HyperD F, Toyopearl SP-650, and Toyopearl MegaCap II SP-550EC). A conditioning step was introduced in order to reduce the urea concentration prior to the refolding step. Refolding was initiated by applying an elution buffer containing a high concentration of Tris-HCl plus common refolding additives. The actual refolding process occurred concurrently with the elution step and was completed in the collected fraction. With Capto S, POROS HS, and Fractogel SO(3)(-), refolding could be performed at column loadings of 50mg fusion protein/ml gel, resulting in a final eluate concentration of around 10-15 mg/ml, with refolding and cleavage step yields of around 75%. The overall yield of recovered peptide reached 50%. Similar yields were obtained using the anion exchange system and the pep6His fusion peptide. This chromatographic refolding process allows processing of fusion peptides at a concentration range 10- to 100-fold higher than that observed for common refolding systems.