Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli.

Recombinant human erythropoietin produced by mammalian cells contains about 40% carbohydrates which maintain its stability and long residence in body. However, mammalian derived Epo has low yields and high costs of production. In this article, a cost-effective strategy of producing non-glycosylated Epo from Escherichia coli and then PEGylating it to replace the role of sugar chains was investigated.

[Molecular imprinting-flow injection chemiluminescence method for determination of doxycycline hydrochloride].

Doxycycline hydrochloride can enhance the chemiluminescence of potassium ferricyanide and luminol in alkaline medium. So a molecular imprinting-flow-injection chemiluminescence method for the determination of doxycycline hydrochloride was established by using doxycycline hydrochloride-imprinted polymers as recognition material and potassium ferricyanide and luminol as detection system. Doxycycline hydrochloride-imprinted polymer was synthesized using methacrylic acid as functional monomer and ethylene glycol dimethacrylate as cross-linker.

[Investigation on degradation process of collagens during manufacturing procedure of E'jiao].

Objective: To investigate the degradation process of collagens and identify the key unit operation during manufacturing process of E'jiao.

Method: Samples in different unit operations were withdrawn, and their amino acid compositions and the molecular weight ranges were determined. The peptide composition was analyzed by high-performance liquid chromatography/mass spectrometry.

A novel stationary phase derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed protein chromatography.

Using agarose coated gigaporous polystyrene microspheres as a base support, a novel anion exchanger (DEAE-AP) has been developed after functionalization with diethylaminoethyl chloride. The gigaporous structure, static adsorption behavior, and chromatographic properties of DEAE-AP medium were characterized and compared with those of commercially available resin DEAE Sepharose Fast Flow (DEAE-FF). The results implied that there existed some through pores in DEAE-AP microspheres, which effectively reduced resistance to stagnant mobile phase mass transfer by inducing convective flow of mobile phase in the gigapores of medium.

Poly (ethylene glycol) prodrug for anthracyclines via N-Mannich base linker: design, synthesis and biological evaluation.

Poly (ethylene glycol)s (PEGs) are potential drug carriers for improving the therapeutic index of anticancer agents. In this work, a novel methodology for constructing PEG prodrug of anthracycline anticancer drugs was developed based on N-Mannich base of salicylamide and its 2-acyloxymethylated derivative. The resultant conjugates first subjected to in vitro hydrolysis testing, which revealed the release behavior of newly synthesized PEG prodrugs could be adjusted by the status of 2-hydroxy group of salicylamide.

Solid phase pegylation of hemoglobin.

A solid phase conjugation process was developed for attachment of polyethylene glycol to hemoglobin molecule. Bovine hemoglobin was loaded onto an ion exchange chromatography column and adsorbed by the solid medium. Succinimidyl carbonate mPEG was introduced in the mobile phase after the adsorption.

Hollow quaternized chitosan microspheres increase the therapeutic effect of orally administered insulin.

The delivery of insulin by non-parenteral routes has gained significant attention over the last two decades. In the present study, we prepared hollow quaternized chitosan microspheres by the SPG membrane emulsification technique and glutaraldehyde cross-linking method. The structural properties, as well as the uniform size and autofluorescence, enabled us to develop oral delivery of insulin which conserved the bioactivity of the encapsulated insulin, achieving bioadhesion of microspheres, increasing the loading ability and optimizing the release profile.

Enhanced circulation half-life of site-specific PEGylated rhG-CSF: optimization of PEG molecular weight.

Recombinant human granulocyte colony stimulating factor (rhG-CSF) and its PEGylated product "mono-PEG20-GCSF" have already been widely used for treatment of all kinds of neutropenia. However, the high required dosage of mono-PEG20-GCSF made it relatively expensive in clinical use. We postulated that an N-terminal site-specific PEGylated rhG-CSF with higher PEG Mw (PEG30 kDa) might be able to achieve longer circulation half-life while retaining its bioactivity, allowing the reduction of dosage for clinical use.

Separation of five isomers of dihydroxybenzoic acid by high-speed counter-current chromatography with dual-rotation elution method.

Five small molecular isomers, 2,3-, 2,4-, 2,6-, 3,4-, and 3,5-dihydroxybenzoic acid, were successfully separated in one step with solvent system n-hexane-ethyl acetate-methanol-water (1:5:1.5:5) on high-speed counter-current chromatography (HSCCC). A new method, dual-rotation elution, was successfully used to decrease separation time and increase resolution.

A solid-phase adsorption method for PEGylation of human serum albumin and staphylokinase: preparation, purification and biochemical characterization.

A solid-phase adsorption method was developed to circumvent the disadvantage of the conventional liquid-phase PEGylation, i.e. the heterogeneity of the PEGylated products.

Cooperative effects of urea and L-arginine on protein refolding.

The use of low concentrations of urea, guanidinium chloride or arginine has been reported in the literature to increase protein refolding and yield of active proteins by suppressing aggregate formation. However, no studies have yet examined whether these substances can exert synergistic or cooperative effects when used in combination. In this work, a comparative study was carried out on refolding of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in the presence of different concentrations of urea, guanidinium chloride or arginine.

Double emulsion templated microcapsules with single hollow cavities and thickness-controllable shells.

A novel form of microcapsules, which possess single hollow cavities and thickness-controllable shells, were prepared by a two-step emulsification, emulsion ripening, and suspension polymerization. Parameters on morphology control of water-in-oil-in-water (W/O/W) emulsion globules were particularly investigated in this study, and a universal strategy to prepare single-core water-in-oil (W/O) globules from their multicore precursors was proposed. These single-core globules were further utilized as templates for solid microcapsules by the suspension polymerization, during which the phase-separation mechanism could be employed to form nanochannels across the shells.

Different effects of L-arginine on protein refolding: suppressing aggregates of hydrophobic interaction, not covalent binding.

Arginine is one of the most favorable additives in protein refolding. However, arginine does not work for certain disulfide-bond-containing proteins, which is not yet well explained. In this work, refolding of three proteins in the presence of 0-2 M arginine was investigated and compared.

Glycerol-assisted hydrophobic interaction chromatography improving refolding of recombinant human granulocyte colony-stimulating factor.

Efficient refolding of recombinant proteins in the forms of inclusion bodies at higher concentration remains challenging. Here, we report a strategy of a dual-gradient hydrophobic interaction chromatography (HIC) mode to refold recombinant human granulocyte colony-stimulating factor from its inclusion bodies at high protein concentration. The strategy was taken to meet the demand of dynamic refolding proceeding by gradually decrease the denaturant (guanidine-HCl) concentration and gradually increase the hydrophilicity of media (column of Poros PE 20) with glycerol as additive to provide a mild refolding surroundings.

[Interferon alpha-2b modified with polyethylene glycol].

In order to obtain a more stable PEGylated interferon alpha-2b, and prolong its half life, interferon alpha-2b (IFN alpha-2b) was modified with monomethoxy polyethylene glycol propionaldehyde (mPEG-ALD) 20000. It was found that the optimized reaction condition for the maximum bioactivity and highest PEGylation degree of the mono PEGylated interferon alpha-2b was as follows: in 20 mmol/L, pH 6.5, citric acid and sodium dihydrogen phosphate buffer, the concentration of IFN alpha-2b was 4 mg/mL, and the molar ratio of PEG/IFN alpha-2b was 8:1, and the reaction time was 20 h at 4 degrees C.

An effective way to hydrophilize gigaporous polystyrene microspheres as rapid chromatographic separation media for proteins.

To overcome the disadvantages of protein denaturation and nonspecific adsorption on poly(styrene-divinylbenzene) (PS) medium as a chromatographic support, gigaporous PS microspheres prepared in our previous study were coated with hydrophobically modified agarose (phenoxyl agarose, Agap). Both the modification of agarose and the gigaporous structure of PS microspheres provided an advantage that facilitated the coating of Agap onto PS microspheres. The amount of Agap adsorbed onto the PS surface was examined as a function of the polymer concentration, and various samples of microspheres, differing in surface Agap density, were prepared.

Preparation of hierarchical hollow CaCO3 particles and the application as anticancer drug carrier.

One-pot approach to couple the crystallization of CaCO(3) nanoparticles and the in situ symmetry-breaking assembly of these crystallites into hollow spherical shells was developed under the templating effect of a soluble starch. Further functional study using HP-a as an anticancer drug carrier (DOX) demonstrated its advantages for localizing drug release by the pH value-sensitive structure and enhancing cytotoxicity by increasing cellular uptake, perinuclear accumulation, and nuclear entry.

[Effect of solution environment on the purification of pertussis toxin].

The low recovery of pertussis toxin (PT) and the low resolving efficiency of chromatography, due to the instability of PT in low salt condition, are the main challenges for its purification. We aplied 2 mol/L urea to prevent the aggregation and disassociation of PT during the purification by ion-exchange chromatography (IEC) and gel filtration chromatography (GFC). The effect of urea on the purification of PT was studied by ELISA assay and non-reduced SDS-PAGE.

[Primary structure determination of hirudin and reteplase fusion protein by LC/ESI-MS/MS spectrometry].

The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS.

pH neutralization while succinic acid adsorption onto anion-exchange resins.

Succinic acid is a useful chemical and its purification from fermentation broth by ion-exchange resins has widely drawn attention. In this study, pH neutralization in the process of adsorption of succinic acid from model solutions and fermentation broth by anion-exchange resin NERCB 04 has been tested. Adsorption capacity of NERCB 04 was about 0.

Thermosensitive polymer-conjugated albumin nanospheres as thermal targeting anti-cancer drug carrier.

Thermosensitive Poly(N-isopropylacrylamide-co-acrylamide-co-allylamine) (PNIPAM-AAm-AA)-conjugated albumin nanospheres (PAN) was developed as a new thermal targeting anti-cancer drug carrier by conjugating PNIPAM-AAm-AA on the surface of albumin nanospheres (AN). AN with diameter below 200nm and narrow size distribution was successfully prepared in the first step with desolvation technique. PNIPAM-AAm-AA with different molecular weight (M(w)) was synthesized in the second step by radical polymerization and conjugated onto the surface of AN.

Preparation of uniform-sized PELA microspheres with high encapsulation efficiency of antigen by premix membrane emulsification.

Relatively uniform-sized poly(lactide-co-ethylene glycol) (PELA) microspheres with high encapsulation efficiency were prepared rapidly by a novel method combining emulsion-solvent extraction and premix membrane emulsification. Briefly, preparation of coarse double emulsions was followed by additional premix membrane emulsification, and antigen-loaded microspheres were obtained by further solidification. Under the optimum condition, the particle size was about 1 mum and the coefficient of variation (CV) value was 18.

Uniform-sized PLA nanoparticles: preparation by premix membrane emulsification.

Nanoparticle size is crucial to drug release behavior and biodistribution in vivo, but few studies have been performed on biodegradable nanoparticles with narrow size distribution. In this note, uniform-sized nanoparticles were prepared by a facile method combining emulsion-solvent removal and premix membrane emulsification for the first time. After preparation of coarse emulsions, additional premix membrane emulsification with very high pressure was occupied to achieve uniform-sized nanodroplets, and nanoparticles were formed by further solidification.

Porogen effects in synthesis of uniform micrometer-sized poly(divinylbenzene) microspheres with high surface areas.

A new method of synthesizing uniform poly(divinylbenzene) (polyDVB) microspheres with high specific surface areas was designed by combining Shirasu porous glass (SPG) membrane emulsification, suspension polymerization, and post-crosslinking techniques. It was shown that the physicochemical properties of porogens have a great influence on the size distribution and porous features of microspheres. The low aqueous solubility of porogen facilitated preparation of uniform emulsions and microspheres, and high aqueous solubility led to polydispersed emulsions and poor microsphere yields.

Bioprocess of uniform-sized crosslinked chitosan microspheres in rats following oral administration.

Chitosan microspheres have a great potential in pharmaceutical application. In this study, uniform-sized chitosan microspheres crosslinked with glutaraldehyde (CG microspheres) were prepared by Shirasu Porous Glass (SPG) membrane emulsification technique. Based on the characterizations of uniform size and autofluorescence, it was possible to develop a new detecting system for observing and quantifying the CG microspheres in rats with three different diameters (2.