Scratch tests were performed with fish juice containing high and low molecular weight compounds obtained by ultrafiltration and with degradation compounds known to accumulate in fish stored on ice. ...75 volunteers were tested. The peptide pattern in raw fish juice and its high and low molecular weight fractions were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the concentration of protein in the various fractions was determined. Fish products were analysed for bacteria and algae and the concentration of degradation compounds was measured. Mainly high molecular weight compounds (polypeptides) of fish juice were found to be responsible for the skin symptoms.
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Liquid-liquid phase separation (LLPS) has long been observed during the physical stability investigation of therapeutic protein formulations. The buffer conditions and the presence of ...various excipients are thought to play important roles in the formulation development of monoclonal antibodies (mAbs). In this study, the effects of several small-molecule excipients (histidine, alanine, glycine, sodium phosphate, sodium chloride, sorbitol and sucrose) with diverse physical-chemical properties on LLPS of a model IgG1 (JM2) solutions were investigated by multiple techniques, including UV–vis spectroscopy, circular dichroism, differential scanning calorimetry/fluorimetry, size exclusion chromatography and dynamic light scattering. The LLPS of JM2 was confirmed to be a thermodynamic equilibrium process with no structural changes or irreversible aggregation of proteins. Phase diagrams of various JM2 formulations were constructed, suggesting that the phase behavior of JM2 was dependent on the solution pH, ionic strength and the presence of other excipients such as glycine, alanine, sorbitol and sucrose. Furthermore, we demonstrated that for this mAb, the interaction parameter (kD) determined at low protein concentration appeared to be a good predictor for the occurrence of LLPS at high concentration.
The present study reports a method to determine the total protein concentration or concentration of a protein of interest in a protein–protein conjugate using ultraviolet absorption, after ...determining the molar ratio of proteins in the conjugates, from which an extinction coefficient can be calculated. A Microsoft Excel solver-based template using amino acid analysis data was developed for determining the molar ratio. The percent mass of each protein in the conjugate is calculated from the amino acid composition data using the least squares method in the Microsoft Excel solver function, and the percent mass is converted to molar portion of each protein using corresponding molecular weight. A molar ratio is obtained by dividing the molar portion of protein 1 by the molar portion of protein 2. A weighted extinction coefficient is calculated using the molar ratio, and the total protein concentration is determined using ultraviolet absorption at 280nm. The accuracy of the method was verified using mixtures of known proteins. The present study provides a rapid, simple and accurate method for determining protein concentration in protein–protein conjugates.
An ultracentrifugation procedure is described to concentrate protein solutions on the microliter scale. Protein solutions were centrifuged in U-shaped lengths of polyethylene tubing at 160000 X g for ...15 h and thereafter fractionated by cutting the tubing. The method can be performed in a conventional ultracentrifuge and needs no special equipment.
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