Preparation and Characterization of Site-Specific Fatty Chain-Modified Recombinant Human Granulocyte Colony Stimulating Factor.

The clinical use of recombinant human granulocyte colony-stimulating factor (rhG-CSF) is limited by its short serum half-life. In this study, a long-acting strategy for site-specific modification of rhG-CSF with 1-pentadecyl-1H-pyrrole-2,5-dione (C15 fatty chain-maleimide, C15-MAL) was studied in mixed DMSO-aqueous solutions. The factors influencing the conjugation reaction were investigated and optimized, and a high yield of the desired product (C15-rhG-CSF) was achieved. Subsequently, C15-rhG-CSF product was efficiently purified using preparative liquid chromatography, and further characterized. Circular dichroism spectroscopy analysis showed that the secondary structure of C15-rhG-CSF had no significant difference from unmodified rhG-CSF. C15-rhG-CSF retained 87.2% of in vitro bioactivity of unmodified rhG-CSF. The pharmacokinetic study showed that the serum half-life of C15-rhG-CSF in mice was 2.08-fold longer than that of unmodified rhG-CSF. Furthermore, C15-rhG-CSF by single-dose subcutaneous administration showed better in vivo efficacy than those of both PEG10k-rhG-CSF by single-dose administration and rhG-CSF by multiple doses administration. This study demonstrated the potential of C15-rhG-CSF being developed into a novel drug candidate as well as an efficient process for the development of long-acting protein and peptide drugs.

Study of the fibrinolytic activity of serrapeptase and its in vitro thrombolytic effects

Abstract Serrapeptase, a proteolytic enzyme, has been used for the adjuvant treatment of many diseases. However, its fibrinolytic activity is still uncertain. Herein, the fibrinolytic activity of serrapeptase and its in vitro thrombolytic effects were investigated. The results showed that the fibrinolytic activity of serrapeptase was 1295 U/mg, and the specific activity was 3867 U/mg of protein when its proteolytic activity toward casein was 2800 U/mg. The optimum temperature and pH for serrapeptase activity were 37-40°C and 9.0, respectively. At 1 mmol/L, Zn2+, Mn2+ and Fe2+ could activate the fibrinolytic activity of serrapeptase, while K+, Cu2+, sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) inhibited it. In vitro tests showed that serrapeptase could completely prevent blood coagulation at 150 U/mL, and the percentage of blood clot lysis reached 96.6% at 37°C after 4 h at 300 U/mL. These results indicate that serrapeptase has excellent fibrinolytic activity, and can be used as a health food or candidate drug for the prevention or treatment of thrombotic diseases.

Simultaneous solid-liquid separation and primary purification of clavulanic acid from fermentation broth of Streptomyces clavuligerus using salting-out extraction system.

Clavulanic acid (CA) is usually used together with other ß-lactam antibiotics as combination drugs to inhibit bacterial ß-lactamases, which is mainly produced from the fermentation of microorganism such as Streptomyces clavuligerus. Recently, it is still a challenge for downstream processing of low concentration and unstable CA from fermentation broth with high solid content, high viscosity, and small cell size. In this study, an integrated process was developed for simultaneous solid-liquid separation and primary purification of CA from real fermentation broth of S. clavuligerus using salting-out extraction system (SOES). First, different SOESs were investigated, and a suitable SOES composed of ethanol/phosphate was chosen and further optimized using the pretreated fermentation broth. Then, the optimal system composed of 20% ethanol/15% K2HPO4 and 10% KH2PO4 w/w was used to direct separation of CA from untreated fermentation broth. The result showed that the partition coefficient (K) and recovery yield (Y) of CA from untreated fermentation broth were 29.13 and 96.8%, respectively. Simultaneously, the removal rates of the cells and proteins were 99.8% and 63.3%, respectively. Compared with the traditional method of membrane filtration or liquid-liquid extraction system, this developed SOES showed the advantages of simple operation, shorter operation time, lower process cost and higher recovery yield of CA. These results demonstrated that the developed SOES could be used as an attractive alternative for the downstream processing of CA from real fermentation broth.

Selective carboxypropionylation of chitosan: synthesis, characterization, blood compatibility, and degradation.

Two water-soluble chitosan (WSC) derivatives of N-succinyl-chitosan (NSCS) and N,O-succinyl-chitosan (NOSCS) with a degree of substitution (DS) that ranged form 0.28 to 0.61 were selectively synthesized by varying the molar ration of succinic anhydride and chitosan. The chemical structure and physical properties of the chitosan derivatives were characterized by FT-IR, 1H NMR, and XRD. XRD analysis showed that the derivatives were amorphous. The lysozyme enzymatic degradation results revealed that the NSCS was of higher susceptibility to lysozyme. The degradation rate and the solubility of the chitosan derivatives were strongly determined by the degree of substitution and the position of the substitution. The results of antithrombotic properties, hemolytic properties and anticoagulant properties of WSCs indicated that the blood compatibility was dramatically improved, and the carboxyl group introduced on the C-6 or C-2 hydroxyl group appeared to impact anticoagulant activity in different ways.

Separation of cytidine 5'-triphosphate biosynthesized from cytidine 5'-monophosphate on ion-exchange resin and HPLC analysis of cytidine compounds.

Conditions were studied in the biosynthesis of cytidine 5'-triphosphate (CTP) from cytidine 5'-monophosphate (CMP). A 201 x 7 anion ion-exchange resin was applied for the separation of CTP from CMP. Adsorption isotherm and elution conditions (eluant, eluant concentration, flow rate, sample volume loaded) were investigated. At the same time, a new high-performance liquid chromatography on an anion ion-exchange column WAX-1 with UV detector at 260 nm was developed to measure CMP, cytidine 5'-diphosphate (CDP), and CTP. The retention time for CMP, CDP, and CTP are 0.723, 1.448, and 4.432 min, respectively. This new rapid high-performance liquid chromatography (HPLC) method for the analysis of cytidine compounds in biological sample has a wide linear range with high precision and repeatability.