Using chemical chaperones to increase recombinant human erythropoietin secretion in CHO cell line.

In recombinant protein production, over-expressed genes induce unfolded protein response (UPR), overloaded protein aggregation in endoplasmic reticulum and its expansion. In this study, we have used 16 chemicals to improve erythropoietin production in engineered CHO cells and tried to study the mechanism of reducing protein aggregation in each treatment. Endoplasmic reticulum expansion was studied through endoplasmic reticulum specific labeling with utilizing fluorescent glibenclamide and its molecular chaperones expression were studied by real-time polymerase chain reaction. The increase in the mRNA level of EPO and endoplasmic reticulum chaperones GRP78/BiP, XBP1, ATF6, and ATF4 in different chemical treatments were not related to ER expansion. On the other hand, ER expansion in beta alanine, beta cyclodextrin and taurine treatments resulted in increased EPO secretion. Dramatically increase in EPO expression in conjugated linoleic acid, spermidine, trehalose, and maltose (19, 20, 16, and 19-fold, respectively) did not increase erythropoietin productivity, but betaine which did not caused ER expansion, with minor increase in EPO gene expression increase EPO productivity. The results indicated that betaine increase EPO secretion in engineered CHO cell line without relation to ER expansion and molecular chaperones expression.

Physicochemical screening for chemical stabilizer of erythropoietin to prevent its aggregation.

Recombinant protein aggregation is a problematic issue and can provoke immunological response. The aim of this study was to analyze the stability of erythropoietin (EPO), as a therapeutic protein expressed in mammalian cells, in the presence of different chemicals and find a specific stabilizer for EPO. The effects of several chemicals, including mannitol, betaine, trehalose, taurine, linoleic acid, beta-cyclodextrin, copper sulfate, spermidine, maltose, maltodextrin, sucrose, dextran, beta-alanine, myo-inositol, and cysteine, on protein stabilization through the thermally induced aggregation of EPO were monitored. Based on the results of turbidity assay for thermal aggregation, three different patterns were observed for protein stability of active pharmaceutical ingredient of EPO, namely, accelerated, dose-dependent, and inhibitory behaviors for aggregate formation due to treatment with spermidine, mannitol, and betaine, respectively. According to circular dichroism outcomes, EPO treatment with betaine and spermidine resulted in different helical contents of the secondary structure. Dynamic light scattering experiments indicated that treating EPO with betaine resulted in less protein aggregation due to freeze and thaw stresses. Betaine was able to stabilize EPO and inhibit its aggregation, as opposed to spermidine that induced protein aggregation.

An investigation into the parameters affecting preparation of polybutyl cyanoacrylate nanoparticles by emulsion polymerization.

Emulsion polymerization was used to synthesize poly butyl cyanoacrylate nanoparticles in presence of steric stabilizer dextran 70. Nanoparticles were characterized by particle size analysis, scanning electron microscopy and light microscopy. Polymerization factors affecting particle size and distribution such as dextran 70, polysorbate 80 (PS 80) and H(+) concentration, polymerization time and temperature, and sonication were studied. Distinct concentrations of stabilizer were needed to produce proper nanoparticles. In this case, the appropriate value was 2 % of total volume. At pH 4 significant decrease in production efficiency demonstrated the substantial effect of H(+) concentration on nanoparticles. Furthermore significant increases in particle size and distribution was observed at 50 °C compared to room temperature. 0.001 % (v/v) PS 80 represented notable influence on size and distribution. In addition, shaped nanoparticles were obtained by altering polymerization time from 5.5 h to 18 h. Finally, nanoparticle features were influenced by different factors. Appropriate manipulating of such factors can lead to obtaining desirable nanoparticles.

Improvement of Desferrioxamine B Production of Streptomyces pilosus ATCC 19797 With Use of Protease Inhibitor and Minerals Related to Its Activity.

The relationship between protease and Desferal production was assayed. Experiments were performed using a cultivation of Streptomyces pilosus ATCC 19797 in soybean broth medium containing 2% soybean flour and 2% mannitol. The metabolism of the trihydroxamic acid sidrophore desferrioxamine B and protease production by a S. pilosus in nine continues days after culture were investigated as well as the effect of protease inhibitors was examined. It is found that the Desferal formation decreased with increased protease production. Also the effect of protease inhibitors and minerals in determined day of protease production in the culture medium by S. pilosus has been investigated.

Production and Purification of Rabbit's Polyclonal Antibody Against Factor VIII.

The attempt is made to produce recombinant factor VIII but the first step in producing such product is production and purification of rabbit's polyclonal antibody against factor VIII. The second and third steps involve monoclonal antibody and recombinant factor VIII production. Factor VIII is one of the most important coagulating factor where its deficiency leads to diseases like hemophilia type A or classic. It is an inherited disease. Previously, it was obtained through fractionation of blood plasma of blood donors. After processing, factor VIII could be used to manage such patients. Due to transfer of viral disease like hepatitis and HIV through factor VIII obtained by fractionation, high cost of production, insufficiency of the donors and the process of virus removal, thus production of factor VIII through recombinant technology can be useful and helpful. The reaction between antibody and antigen is one the most specific reaction; therefore, such reaction can be employed to identify factor VIII. Thereby, rabbits were injected several times with adjuvant-linked antigen to produce antibody. The antibody was separated from the blood sample, purified and used to identify factor VIII in the research.