The Effect of Arginine as an Anti-Aggregation Excipient on Recombinant Human Growth Hormone

Aggregation is one of the main physical instabilities of proteins, which might occur during all steps of the manufacturing and storage of products. The presence of protein aggregates may result in the reduction of activity, induce immunologic responses and failure of therapeutic efficiency. Therefore, using additives in drug formulations is one of the essential approaches to prevent protein aggregation. The main objective of this study was to evaluate the inhibitory influence of arginine or glycine as excipients on the aggregation behavior of recombinant human growth hormone (rhGH). Two types of mechanical and thermal stresses including freeze-thaw and vortex-agitation were applied to the 1 mg/mL protein solution in PBS buffer (25 mM, pH = 7) in the presence and absence of arginine and glycine. The influence of arginine or glycine at the concentration of 320 mM on reduction of rhGH thermal/mechanical-induced aggregation was evaluated using SE-HPLC and turbidity measurement. The results of this study revealed that the monomer concentration decreased linearly; and therefore, aggregate formation was intensified with the increase in the number of freeze-thaw cycles. Moreover, it was found that a significant amount of rhGH (> 80%) was rapidly adsorbed at the walls of the vessels or converted to insoluble aggregates. Arginine decreased the insoluble aggregate formed during the freeze-thaw cycling more effectively than glycine. In addition, following the vortex-agitation stress, arginine had the optimum preventive effect in aggregate formation in contrast to glycine, which increased the formation of insoluble aggregates. The findings revealed that arginine may be a potential additive in preserving rhGH against thermal/mechanical-induced aggregation.Highlights Aggregation is one of the main physical instabilities of proteins.Protein aggregation may result in a compromise of safety and efficacy of biopharmaceutical products.Arginine at the concentration of 320 mM reduced rhGH thermal/mechanical-induced aggregation. Arginine, in contrast to glycine, optimally decreased the formation of insoluble aggregates

Optimization of single-walled carbon nanotube solubility by noncovalent PEGylation using experimental design methods

In this study, noncovalent functionalization of single-walled carbon nanotubes (SWCNTs) with phospholipid-polyethylene glycols (Pl-PEGs) was performed to improve the solubility of SWCNTs in aqueous solution. Two kinds of PEG derivatives, ie, Pl-PEG 2000 and Pl-PEG 5000, were used for the PEGylation process. An experimental design technique (D-optimal design and second-order polynomial equations) was applied to investigate the effect of variables on PEGylation and the solubility of SWCNTs. The type of PEG derivative was selected as a qualitative parameter, and the PEG/SWCNT weight ratio and sonication time were applied as quantitative variables for the experimental design. Optimization was performed for two responses, aqueous solubility and loading efficiency. The grafting of PEG to the carbon nanostructure was determined by thermogravimetric analysis, Raman spectroscopy, and scanning electron microscopy. Aqueous solubility and loading efficiency were determined by ultraviolet-visible spectrophotometry and measurement of free amine groups, respectively. Results showed that Pl-PEGs were grafted onto SWCNTs. Aqueous solubility of 0.84 mg/mL and loading efficiency of nearly 98% were achieved for the prepared Pl-PEG 5000-SWCNT conjugates. Evaluation of functionalized SWCNTs showed that our noncovalent functionalization protocol could considerably increase aqueous solubility, which is an essential criterion in the design of a carbon nanotube-based drug delivery system and its biodistribution

Direct Immobilization of Coagulation Factor VIII on Au/Fe3O4 Shell/Core Magnetic Nanoparticles for Analytical Application

Protein-coated nanoparticles have diverse applications in biomedical science. The protein hydrophobic domains or surface electrostatic charge conducts adsorption of proteins to different surfaces. This property can be customized to immobilize specific molecules on solid supports for experimental screenings or purification processes. To develop highly selective affinity ligands—such as aptamers—against specific protein targets, protein-coated magnetic particles have been successfully applied. This approach could be highly efficient in affinity ligand development against coagulation factor VIII.In this study, magnetic nanoparticles were prepared by co-precipitation method and, then, a gold coating was run on the MNPs’ surface. The gold coating could add some attractive specifications to the protein immobilized nanoparticles during the aptamer selection process, such as simultaneous affinity determination of aptameric oligonucleotides by fluorescence-based methods. The gold surface has been indicated as a specific feature for covalent binding to the sulphur functional groups of various molecules. In proteins, sulphur units of cysteine or methionine might be bound covalently to the gold surface. In addition, nonspecific and non-covalent attachment of proteins to the gold particles may be performed. Therefore, a series of samples containing different mass ratios of protein to gold magnetic nanoparticles (GMNPs) were evaluated to find the best conditions for coagulation factor VIII immobilization. The results showed that the best condition for high coating efficiency was 48 h incubation at 4 ºC of protein and GMNPs with a mass ratio of 0.5% in PBS 25mM, with pH=7 as binding buffer.Highlights:Magnetic nanoparticles are the most attractive nanostructures in biomedical and bio-analytical fields.The protein coating on MNPs has been found to have wide clinical and analytical applications.Coagulation factor VIII (FVIII) is a valuable therapeutic human protein in the market.Attachment of a large protein like F VIII to GMNPs is affected by various environmental factors

Immobilization of Laccase in Alginate-Gelatin Mixed Gel and Decolorization of Synthetic Dyes

Alginate-gelatin mixed gel was applied to immobilized laccase for decolorization of some synthetic dyes including crystal violet. The immobilization procedure was accomplished by adding alginate to a gelatin solution containing the enzyme and the subsequent dropwise addition of the mixture into a stirred CaCl2 solution. The obtained data showed that both immobilized and free enzymes acted optimally at 50°C for removal of crystal violet, but the entrapped enzyme showed higher thermal stability compared to the free enzyme. The immobilized enzyme represented optimum decolorization at pH 8. Reusability of the entrapped laccase was also studied and the results showed that ca. 85% activity was retained after five successive cycles. The best removal condition was applied for decolorization of seven other synthetic dyes. Results showed that the maximum and minimum dye removal was related to amido black 10B and eosin, respectively

Green synthesis of gold nanoparticles by the marine microalga Tetraselmis suecica

The application of green-synthesis principles is one of the most impressive research fields for the production of nanoparticles. Different kinds of biological systems have been used for this purpose. In the present study, AuNPs (gold nanoparticles) were prepared within a short time period using a fresh cell extract of the marine microalga Tetraselmis suecica as a reducing agent of HAuCl4 (chloroauric acid) solution. The UV–visible spectrum of the aqueous medium containing AuNPs indicated a peak at 530 nm, corresponding to the surface plasmon absorbance of AuNPs. The X-ray diffraction pattern also showed a Bragg reflection related to AuNPs. Fourier-transform infrared spectroscopy was performed for analysis of surface functional groups of AuNPs. Transmission electron microscopy and particle-size-distribution patterns determined by the laser-light-scattering method confirmed the formation of well-dispersed AuNPs. The most frequent size of particles was 79 nm

The removal of ρ-chlorophenol in aqueous cultures with free and alginate-immobilized cells of the microalga Tetraselmis suecica

The present study aimed at evaluating the ability of some isolated cyanobacterial and microalgal strains for the removal of ρ-chlorophenol (ρ-CP), an environmentally harmful contaminant. To identify the most efficient species, a screening program was carried out using 15 microalgal and cyanobacterial strains. Among them, Tetraselmis suecica was able to remove 67 % of the ρ-chlorophenol at an initial concentration of 20 mg L−1 from the medium within a 10-day period. The efficacy of the process was dependent on the ρ-chlorophenol concentration. At concentrations above 60 mg L−1 of the pollutant, no removal was observed due to the inhibitory effect of ρ-chlorophenol on the T. suecica cells. The effect of cell immobilization in alginate beads on T. suecica removal capacity was also examined. Using this technique, the removal efficacy for the initial ρ-CP concentration of 20 mg L−1 increased up to 94 %

Laccase Activity in CTAB-Based Water-in-Oil Microemulsions

Abstract The aim of this study was to develop a microemulsion system as a medium for laccasecatalyzed reactions. Phase behavior studies were conducted by constructing partial pseudoternary phase diagrams for systems comprising of cetyltrimethylammonium bromide (CTAB), various organic solvents as the oil phase (i.e., hexane, cyclohexane, heptane, octane, isooctane, toluene, isopropyl myristate), two co-surfactants (i.e., 1-butanol and 1-hexanol) and citrate buffer solution, at various surfactant/co-surfactant weight ratios (R sm ). A monophasic, transparent, non-birefringent area (designated as microemulsion domain) was seen to occur in some phase diagrams along the surfactant/organic solvent axis, the extent of which was dependent mainly upon the nature of co-surfactant and R sm . On each phase diagram, three different water-inoil (w/o) microemulsion systems with less than 50 wt% surfactant mixture and less than 20 wt% of aqueous phase were selected for laccase loading and activity measurements. Results revealed that the catalytic activity of laccase in CTAB-based w/o microemulsions decreased considerably, compared with its activity in the buffer solution, the extent of which depended upon the type of component and their compositions in the microemulsions. It was suggested that the conformational changes due to the electrostatic interactions between the cationic head group of CTAB and the negative enzyme might be the reason for the reduction of laccase activity, once entrapped in the microemulsion