RESUMO
Background: Background: Downstream processing of therapeutic recombinant proteins expressed as the inclusion bodies (IBs) in E. coli is quite challenging. This study aimed to use the quality by design approach for developing the multi-step downstream process of a structurally complex therapeutic Fc-Peptide fusion protein, romiplostim. Methods: Methods: For development of a successful downstream process, risk analysis and experimental designs were used to characterize the most critical quality attributes (CQAs) and effects of process parameters on these quality attributes. Results: Results: The solubilization of IBs was optimized by design of experiment on three parameters with a focus on solubility yield, which resulted in >75% increase of the target protein solubilization. The pH of sample was identified as CQA in anion exchange chromatography that might have an impact on achieving >85% host cell proteins removal and >90% host cell DNA reduction. In the refolding step, process parameters were screened. Cystine/cysteine ratio, pH, and incubation time identified as CPPs were further optimized using Box-Behnken analysis, which >85% of the target protein was refolded. The design space for further purification step by HIC was mapped with a focus on high molecular weight impurities. After polishing by gel filtration, the final product's biological activity showed no statistically significant differences among the groups received romiplostim and Nplate®, as the reference product. Conclusions: Conclusion: This research presents a precise and exhaustive model for mapping the design space in order to describe and anticipate the link between the yield and quality of romiplostim and its downstream process parameters.
Assuntos
Escherichia coli , Corpos de Inclusão , Proteínas Recombinantes de Fusão , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossínteseRESUMO
For biotechnology applications, a novel nanobiocomposite was synthesized based on modification of graphene oxide (GO) by extracted silk fibroin (SF), natural polymer pectin (Pec) and zinc chromite (ZnCr2O4) nanoparticles (NPs). The structure and properties of hybrid nanobiocomposite GO-Pec/SF/ZnCr2O4 such as thermal stability, less toxicity, biocompatibility, antibacterial, and biodegradable were proved by using field emission scanning electron microscope (FE-SEM), Fourier-transformed infrared (FT-IR), Energy dispersive X-ray spectroscopy (EDS), thermal gravimetric analysis (TGA), and X-Ray diffraction (XRD). According to the biological features of substances, the GO-Pec/SF/ZnCr2O4 nanobiocomposite shows perfect results in MTT (83.71 %) and Hemolysis (16.52 %) assays. accordingly, mentioned properties of this nanobiocomposite can be used as a scaffold for medical applications.
Assuntos
Fibroínas , Nanocompostos , Nanopartículas , Óxido de Zinco , Antibacterianos/química , Fibroínas/química , Grafite , Nanocompostos/química , Pectinas , Espectroscopia de Infravermelho com Transformada de Fourier , ZincoRESUMO
ABSTRACT Nanoparticles due to their unique properties have attracted more attention and their bacterial biosynthesis is more favorable because is environmental friendly and the size and yield of nanoparticles can be optimized. The aim of the present study was biosynthesis of Selenium nanoparticles using Bacillus cereus. For this purpose, bacterial culture was prepared in the presence of sodium selenate solution and incubated (30°C, 24 h). The produced nanoparticles were purified through consequent centrifugation, washing with 0.9% NaCl, sonication, washing with Tris- HCl containing Sodium dodecyl sulfate (SDS) and finally isolation with water- octanol two phase systems. Then using Ultraviolet-Visible spectroscopy, dynamic light scattering (DLS), scaning electron microscopy (SEM) and X-ray diffraction (XRD) analysis, nanoparticle production was confirmed. The bioavailability of nanoparticles was also investigated in rat. As a result of this study spherical selenium nanoparticles with a mean diameter of 170 nm were biosynthesized. MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of selenium for Bacillus cereus were same and equal to 75mM. Absorption and secretion of nanoselenium was significantly higher than bulk Selenium (P<0.05). In conclusion in the present study without any chemical substance, spherical Selenium nanoparticles were produced that do not have any environmental contamination. Furthermore, the metabolism of these particles suggests higher absorption rate of them that facilitates its application in medicine and also veterinary medicine.