Dynamic light scattering size exclusion
We present explicit expressions in real space and time for the equilibrium correlation functions of the density and velocity fields in a compressible fluid. Also, numerical results for the motor domains of ncd and kinesin, small arbitrarily shaped proteins with known x-ray structures, show reasonable agreement with the experimental data obtained from transient electric birefringence experiments. This approach has been tested for ellipsoidal particles and a good agreement with theoretical results has been obtained. The average dielectric constant of the mixture is calculated using the finite element method. Since the particles of interest are small compared to the wavelength of light, a quasi-static approximation for the refractive index is used, i.e., that it is equal to the square root of the dielectric constant of the suspension. The form birefringence of the solution is calculated as the difference in its refractive index when all the particles of refractive index n 2 are either parallel to or normal to the direction of the polarization of light. Macromolecules are treated as arbitrarily shaped particles suspended in a solvent of refraction index n 1. In view.ĪBSTRACT An approach based on the finite element method (FEM) is employed to calculate the optical properties of macromolecules, specifically form birefringence. Further, reference will usually be made to these sources, where the original studies are cited. As many comprehensive discussions of the theoretical basis for the methods of interest are available, the limited space here is devoted to some of the more practical aspects of the several scattering methods to be discussed. berry & cotts 1 9/23/97 INTRODUCTION The use of light scattering methods to characterize polymers or colloidal particles in dilute solutions (dispersions) is discussed in the following. The theoretical foundations are summarized to give the expressions most often utilized, central issues in the calibration and use of light scattering photometers are considered, and several examples are discussed, including the use of light scattering as a detector in connection with size exclusion chromatograpy. Owing to its simplicity and low-sample volume requirements, DLS can be used even by hospital pharmacists to confirm absence of protein aggregates immediately before drug administration.Static and dynamic light scattering methods for use in the characterization of dilute solutions of polymers or suspensions of dispersed particles are presented. Although no single techniques can reveal all aspects of protein stability, DLS can serve as a screening tool to detect aggregate formation and cross-validate SE-HPLC results during batch release testing. Results showed that aggregate formation was not detected in some cases by SE-HPLC and the decrease in the concentration of the monomeric forms indicated that such aggregates might have been filtered off the column.
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Generally, good agreement between the results of DLS and SE-HPLC was noted, regardless of the differences in physicochemical properties of the studied biopharmaceuticals. Samples were subjected to forced degradation conditions previously shown to lead to aggregate formation (pH 4.0, 8.0 and 10.0, at 37 ☌ for 24 h) and samples were analyzed using DLS and SE-HPLC. Here, three model biopharmaceutical proteins of different physicochemical properties were selected quadrivalent human papillomavirus virus like particles vaccine (HPV VLP, physically assembled subunit vaccine, 55 kDa), pegylated Interferon (PegIFN, pegylated non-glycosylated protein, 31.3 kDa) and Pegylated Erythropoietin (PegEPO, pegylated and glycosylated protein, 60 kDa). On the other hand, dynamic light scattering (DLS) is a simple and non-destructive technique that can detect high molecular weight physical and chemical aggregates in their native environment. Moreover, low-affinity protein aggregates may dissociate during analysis and thus not detected. However, large protein aggregates may be filtered off and build up on top of the column leading to deterioration in column performance. Size exclusion high performance liquid chromatography (SE-HPLC) has always been the gold standard technique for detection and determination of protein aggregates. Aggregate formation is a major problem affecting both safety and efficacy of biopharmaceuticals and is associated with protein immunogenicity.