Necessary cookies are absolutely essential for the website to function properly. Aggregation is indicated by peaks to the right of those observed in the buffer blank. The correlation between time points is used to determine the radii of nanometre-scale objects in solution.įigure A: DLS data for a compound at aggregating and sub-aggregating concentrations, illustrating the raw correlation functions (left) and the corresponding intensity distribution (right). Preformulation and Formulation Developmentĭynamic light scattering (DLS) is a Biophysical Technique that enables particles to be distinguished on the basis of their size. Sygnature have experience in the utilisation of this technique using the Viscotek DLS reader, to interrogate the stoichiometry of protein-protein interactions and to identify aggregating compounds or non-specific inhibitors, adding value to our client’s discovery programs via hit validation, protein characterisation and assay troubleshooting.ĭLS enables an assessment of compound, protein or protein-ligand binding complex aggregation behaviour by measuring the correlation between the intensity of scattered light over a time course.Fluorescent Thermal Shift Assays (FTSA).This DLS assay offers exceptional convenience, ease-of-use and reproducibility and should be useful for monitoring Adenovector preparations for the formation of aggregates. Ultimately, the entire population can aggregate to such an extent that monomeric particles can no longer be detected in the sample. These changes are also observed in the visual histogram generated by the DLS instrument. During vector particle aggregation, both the Z ave and PDI values significantly increase, leading to the formation of a heterogeneous population of aggregates. This includes both antibody- (polyclonal anti-Adenovirus) and temperature-(37☌) induced viral particle aggregation events. Experimental conditions have also been investigated in order to provide insight into the assay's capability of detecting aggregates. The polydispersity index (PDI), which is an expression of sample homogeneity, is typically observed to be <0.2 in these preparations. Under these optimized conditions, purified Adenovector particles are normally observed to be a monodisperse suspension, having a mean particle size (Z ave) in the range of 94 to 104 nm. Latex microsphere standards are utilized to establish system suitability criteria, thereby assuring assay reproducibility and accuracy. The viscosity and refractive index of the sample matrix are incorporated into the analysis in order to ensure data consistency. This instrument accommodates a wide range of sample concentrations and enables analysis of samples in a temperature-controlled environment. A single cuvette method has been established using a helium-neon laser-based High Performance Particle Sizer (Malvern Instruments HPPS 5001).
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We have developed a method for determining vector particle aggregation that employs dynamic light scattering and enables particle size analysis in the “native” state.
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Conventional biochemical methods for analysis of the viral proteome (SDS-PAGE, Reversed-phase HPLC, MALDI-TOF) are disruptive to the structure and organization of the viral particles and therefore do not provide sufficient insight into the aggregation phenomenon. Modifications that can possibly lead to vector particle aggregation. Gene Editing: Technology & Applications.