Arne Staby

Separation of constituents of fish oil using supercritical fluids: a review of experimental solubility, extraction, and chromatographic data

Abstract The use of supercritical fluids, especially carbon dioxide, in separation of fish oil and derived compounds has increased the last decade due to the expected advantages of supercritical separation processes. In pursuit of the design of supercritical fluid separation processes, phase equilibria and pilot plant data are essential. In this paper we present a survey of literature references of experimental phase equilibria and solubility data on binary and multicomponent systems of supercritical fluids and fish oil or derived compounds, as well as experimental data on supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC). The fish oil related compounds include triglycerides, free fatty acids, fatty acid methyl and ethyl esters, cholesterol, α-tocopherol, phospholipids, squalene etc. The list of references include the experimental pressure and temperature ranges, the number of data points/experiments, and some remarks to the kind of measurements performed and components involved. The literature retrieval includes references to the end of 1992 and a few from 1993.

Novel peptide ligand with high binding capacity for antibody purification

Small synthetic ligands for protein purification have become increasingly interesting with the growing need for cheap chromatographic materials for protein purification and especially for the purification of monoclonal antibodies (mAbs). Today, Protein A-based chromatographic resins are the most commonly used capture step in mAb down stream processing; however, the use of Protein A chromatography is less attractive due to toxic ligand leakage as well as high cost. Whether used as an alternative to the Protein A chromatographic media or as a subsequent polishing step, small synthetic peptide ligands have an advantage over biological ligands; they are cheaper to produce, ligand leakage by enzymatic degradation is either eliminated or significantly reduced, and they can in general better withstand cleaning in place (CIP) conditions such as 0.1M NaOH. Here, we present a novel synthetic peptide ligand for purification of human IgG. Immobilized on WorkBeads, an agarose-based base matrix from Bio-Works, the ligand has a dynamic binding capacity of up to 48 mg/mL and purifies IgG from harvest cell culture fluid with purities and recovery of >93%. The binding affinity is ∼10⁵ M⁻¹ and the interaction is favorable and entropy-driven with an enthalpy penalty. Our results show that the binding of the Fc fragment of IgG is mediated by hydrophobic interactions and that elution at low pH is most likely due to electrostatic repulsion. Furthermore, we have separated aggregated IgG from non-aggregated IgG, indicating that the ligand could be used both as a primary purification step of IgG as well as a subsequent polishing step.

A label‐free methodology for selective protein quantification by means of absorption measurements

The application of high throughput experimentation (HTE) in protein purification process development has created an analytical bottleneck. Using a new label‐free and non‐invasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements, we show that the analytical throughput for selective protein quantification can be increased significantly. An analytical assay based on this new methodology was shown to generate very precise results. Further, the assay was successfully applied as analytics for a resin screening performed in HTE mode. The increase in analytical throughput was obtained without decreasing the level of information when compared to analytical chromatography. This proves its potential as a valuable analytical tool in conjugation with high throughput process development (HTPD). Further, fast selective protein quantification can enhance process control in a commercial production environment and, hence, minimize the need for off‐line release analysis. Biotechnol. Bioeng. 2011;108: 2661–2669.

Mutual solubilities of mono-alcohols and carbon dioxide: A review of experimental data

Abstract A list of references from a literature retrieval presenting experimental mutual solubilities and phase equilibria of binary systems of carbon dioxide and monoalcohols is given. The list includes the pressure and temperature ranges, the number of data points, and the references. The literature retrieval includes references to the middle of 1992.

Phase equilibria of fish oil fatty acid ethyl esters and sub- and supercritical CO2

Abstract Measurements of phase equilibria of fish oil fatty acid ethyl esters in sub- and supercritical carbon dioxide were performed at pressures from 2 to 22 MPa at the temperatures 283.2, 313.2, and 343.2 K. Experimental temperatures, pressures, mass fractions, mole fractions, equilibrium ratios, and densities are reported. The K-values of the esters depend strongly on the system pressure and the number of carbon atoms and have a weaker dependence on the degree of unsaturation and position of the double bonds. The data show that the selectivity is high and the solubility is low at low operating pressure, while at high operating pressure the selectivity is low but the solubility is high. More than 95% of the natural mixture was identified.

How can measurement, monitoring, modeling and control advance cell culture in industrial biotechnology?

This report highlights the potential of measurement, monitoring, modeling and control (M(3) C) methodologies in animal and human cell culture technology. In particular, state-of-the-art of M(3) C technologies and their industrial relevance of existing technology are addressed. It is a summary of an expert panel discussion between biotechnologists and biochemical engineers with both academic and industrial backgrounds. The latest ascents in M(3) C are discussed from a cell culture perspective for industrial process development and production needs. The report concludes with a set of recommendations for targeting M(3) C research toward the industrial interests. These include issues of importance for biotherapeutics production, miniaturization of measurement techniques and modeling methods.

Comparison of loading capacities of various proteins and peptides in culture medium and in pure state.

Chromatographic media suppliers most frequently state the capacities of their gels based on either static capacities or frontal analysis experiments of pure proteins, however, these capacity values are often far from the capacities experienced in the production of such proteins. In this work static and dynamic capacities of various pure industrial proteins or peptides are compared to the capacities of the proteins or peptides under similar conditions in their natural culture medium. The results show a significant decrease in the static and dynamic capacities of the proteins or peptides when present in culture medium due to competitive binding of medium proteins. The proteins and peptides included in this study are: lipolase, glucagon-like peptide-1, truncated prothrombin, insulin precursor, and anti-Factor VII monoclonal antibody.

Measurement of mutual solubilities of 1-Pentanol and supercritical carbon dioxide

Abstract The isothermal phase equilibria of 1-pentanol/carbon dioxide have been measured in a variable volume static cell-type apparatus, at pressures from 2 to 18 MPa at the temperatures of 283.2, 313.2, 343.2, and 373.2 K. Experimental temperatures, pressures, mole fractions, and densities are reported.

Effects of urea induced protein conformational changes on ion exchange chromatographic behavior

Urea is widely employed to facilitate protein separations in ion exchange chromatography at various scales. In this work, five model proteins were used to examine the chromatographic effects of protein conformational changes induced by urea in ion exchange chromatography. Linear gradient experiments were carried out at various urea concentrations and the protein secondary and tertiary structures were evaluated by far UV CD and fluorescence measurements, respectively. The results indicated that chromatographic retention times were well correlated with structural changes and that they were more sensitive to tertiary structural change. Steric Mass Action (SMA) isotherm parameters were also examined and the results indicated that urea induced protein conformational changes could affect both the characteristic charge and equilibrium constants in these systems. Dynamic light scattering analysis of changes in protein size due to urea-induced unfolding indicated that the size of the protein was not correlated with SMA parameter changes. These results indicate that while urea-induced structural changes can have a marked effect on protein chromatographic behavior in IEX, this behavior can be quite complicated and protein specific. These differences in protein behavior may provide insight into how these partially unfolded proteins are interacting with the resin material.

Supercritical fluid chromatographic analysis of fish oils

Various natural and processed fish oil triglyceride mixtures have been analyzed by capillary supercritical fluid chromatography (SFC). The analyses were performed on nonpolar columns to separate the components by lipid class and by the number of carbon atoms. The compounds separated included free fatty acids, squalene, α-tocopherol, cholesterol, wax esters, cholesteryl esters, di- and triglycerides. This kind of analysis is not possible by gas chromatography or high-performance liquid chromatography methods without prior treatment of the fish oil, making SFC superior for this application. Applications of SFC to fish oils are given, including a control analysis of the various process steps in the refining of a fish oil, analysis of a lipase-catalyzed transesterification of a fish oil and the detection of polymeric artifacts.