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Abstract: Recombinant human serum albumin (rHSA) is considered as an alternative of human serum albumin and used to treat patients with severe burn, shock or blood loss. However, separation and purification of rHSA are difficult and have become the bottle neck in industrial production. In this study, ethanol/K2HPO4 aqueous two-phase system (ATPS) and hydrophobic interaction chromatography (HIC) were integrated to provide a new approach for the extraction and purification of rHSA from high density fermentation broth. Using a 0.01–73L ATPS scale, the extraction of rHSA from the fermentation broth attained an average recovery of 100.4%. At the same time, 99.8% of cells and 87.2% of polysaccharides as well as some other protein impurities were also removed. The activity of proteinase A in the broth was also remarkably decreased. The purified rHSA appeared as a single band on reduced SDS–PAGE gel, and it had a purity of 99.1% as determined by HPLC. It was essentially identical to the plasma-derived HSA in terms of molecular weight and circular dichroism spectrum. The total recovery of rHSA was 75.2%, which was 1.1–2.0 times higher than that obtained from conventional processes. Residual polysaccharide was reduced to 1.61μg/mg rHSA and the degree of coloring was lower than that of plasma-derived HSA. The procedure employed in this study has the advantages of simple operation, shorter time, low energy consumption and high yield, and it could produce rHSA with high purity. It is therefore suitable in the production of rHSA and other biological products produced by high-density fermentation. [Copyright &y& Elsevier]

Abstract: The design of gradient simulated moving bed (SMB) chromatographic processes requires an appropriate selection of the chromatographic system followed by the determination of adsorption isotherm parameters in the relevant range of mobile phase conditions. The determination of these parameters can be quite difficult for recombinant target proteins present in complex protein mixtures. The first part of this work includes the estimation of adsorption isotherm parameters for streptokinase and a lumped impurity fraction present in an Escherichia coli cell lysate for a hydrophobic interaction chromatography (HIC) matrix. Perturbation experiments were carried out using a Butyl Sepharose matrix with purified recombinant protein on buffer equilibrated columns as well as with crude cell lysate saturated columns. The Henry constants estimated for streptokinase were found to exhibit in a wide range a linear dependence on the salt concentration in the mobile phase. These parameters were applied in subsequent investigations to design a simulated moving bed (SMB) process capable to purify in a continuous manner recombinant streptokinase from the E. coli cell lysate. [Copyright &y& Elsevier]

Abstract: In the first part of this work adsorption isotherm parameters were acquired to describe the migration of recombinant streptokinase in Butyl Sepharose columns at different salt concentrations. Based on these results, a simulated moving bed (SMB) chromatographic process was designed and realised, which exploits a two-step salt gradient and allows the continuous separation of streptokinase from contaminants present in a clarified Escherichia coli cell lysate solution. This second part describes the design of the three-zone open-loop gradient SMB process applying both equilibrium theory and an equilibrium stage model and presents results of a series of experiments aiming to obtain pure streptokinase. Moreover, the potential of the SMB process and the design approach are evaluated. [Copyright &y& Elsevier]

Abstract: The most significant impurities in plasmid DNA (pDNA)-containing solutions after intermediate recovery are host RNA and genomic DNA. Hydrophobic interaction chromatography (HIC) with ammonium sulfate-based buffers has been used to explore the higher hydrophobicity of those impurities when compared with pDNA. Although successful at lab scale, the large amounts of ammonium sulfate involved constitute an important limitation in terms of waste treatment/disposal if HIC is to be used at process scales. In this work, different HIC ligands (phenyl, butyl, octyl), salts and salt concentrations were screened with the specific goal of replacing ammonium sulfate with an environmentally friendly salt. The combination of phenyl-Sepharose with sodium citrate at high concentrations (≥1.2M) not only allowed the separation of pDNA from RNA, gDNA, endotoxins and protein, but also enabled the separation of supercoiled from open circle isoforms. Alternatively, by exploring negative HIC with the phenyl matrix and 1.0M sodium citrate, it was possible to purify total pDNA from solutions obtained by tangential flow filtration and pre-conditioned with sodium citrate. At the highest loading tested (2mL feed equivalent to ca. 1mg pDNA per mL of bed) despite high-level removal of endotoxins (>98.5%) and proteins (>82%) the purity of recovered plasmid DNA determined by HIC-HPLC was just 73% (primarily due to traces of RNA and gDNA). Much higher purities (close to 100%) were achieved when lower loadings were employed. In summary, this study clearly shows that despite lower purification factors achieved with sodium citrate compared to ammonium sulfate-based HIC, replacing the latter salt with sodium citrate is indeed feasible. [Copyright &y& Elsevier]

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Highlights: [•] Retention behavior of monoclonal IgG1 was investigated on IEC and HIC media. [•] Dynamics of both processes was dominated by kinetic effects. [•] A short-cut method was proposed for mathematical modeling. [•] A dynamic model was used for optimization of the integrated process IEC/HIC. [ABSTRACT FROM AUTHOR]

Highlights: [•] Monoclonal anybody IgG1 was isolated out of a CHO derived supernatant. [•] Different purification techniques were used based on IEC, HIC and ATPE. [•] The best performance was achieved by integrating IEC and HIC. [•] The on-column dilution was used for feed loading in the integrated process. [ABSTRACT FROM AUTHOR]

Abstract: Monosize and magnetic poly(glycidyl methacrylate-N-methacryloyl-(l)-tryptophan) [mPGMATrp] beads (1.6 µm in diameter) were used for hydrophobic affinity capture of lysozyme from chicken egg-white. N-methacryloyl-(l)-tryptophan (MATrp), which gives hydrophobicity to the resulting polymer, was synthesized by reacting methacryloyl chloride and l-tryptophan methyl ester then characterized by Nuclear Magnetic Resonance (NMR). mPGMATrp beads were produced by dispersion polymerization in the presence of magnetite nano-powder. mPGMATrp beads were characterized by means of swelling studies, elemental analysis, Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM). Lysozyme adsorption experiments were performed under different experimental conditions (i.e., lysozyme concentration, temperature, and ionic strength) in magnetically stabilized fluidized bed system, (MSFB). Maximum adsorption capacity was 263.9 mg/g. It was observed that mPGMATrp beads can be used without significant loss in lysozyme adsorption capacity after 25 adsorption-elution cycle. [Copyright &y& Elsevier]