Measuring Protein Mobility with Electrophoretic Light Scattering

The author describes a method to avoid protein aggregation when using light scattering systems.
May 01, 2013
Volume 26, Issue 5

Stephen Ball
As the number of biopharmaceutical formulations that use proteins as drug molecules continues to increase, the spotlight is being directed on the analytical methods used in their development, formulation, and quality control. Protein mobility is one property that has been identified as a promising indicator of formulation stability, viscosity, and behavior. This article describes an approach to using electrophoretic light scattering to make protein mobility measurements.

Light scattering techniques are widely used in protein characterization. Dynamic light scattering (DLS) is established in the measurement of particle and molecular size, and in studying the interactions between proteins. Electrophoretic light scattering (ELS) is used to measure the electrophoretic mobility of particles in dispersion or molecules (such as proteins) in solution. This mobility is often converted into zeta potential to enable comparison of materials under different conditions. In the case of proteins, the measurement of protein mobility allows the calculation of protein charge, which in turn relates to factors such as activity and reaction kinetics. Recent advances in instrumentation and methodologies are addressing the technical challenges of using light scattering to make mobility measurements on proteins.


Martin McCarthy/Getty Images; Dan Ward
The fundamental physical principle in ELS is that of electrophoresis. A sample is introduced into a cell containing two electrodes. An electrical field is applied and particles or molecules that have a net charge, or more strictly a net zeta potential, will migrate towards the oppositely charged electrode with a velocity, known as the electrophoretic mobility, that is related to their zeta potential. This velocity is measured using the laser Doppler technique, where the frequency shift or phase shift of an incident laser beam, caused by the moving particles, is measured as the particle mobility.

Experimentally, protein mobility measurements present two practical challenges. First, working with protein solutions often means working with dilute concentrations, low DLS count rates, and low electrophoretic mobilities. Second, the act of applying an electric field to the sample can damage the protein by stimulating aggregation, with resultant mobility measurements reflecting the aggregate molecules rather than the native protein.

A new approach combines a high sensitivity light scattering system (Zetasizer Nano ZSP, Malvern Instruments) with a diffusion barrier technique that separates the molecules in the sample from the electrodes, to avoid the risk of aggregation. A measurement protocol regulates voltage and temperature; and automatic size measurements before and after the electrophoretic mobility measurement verify that no aggregation has occurred.

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