Electrical charge is a fundamental property of all macromolecules. In colloidal suspensions, the amount of charge and screening developed at the interfaces between particles and media is of utmost importance in determining the formulation stability. For many biomolecules such as proteins, electrostatic interactions also exercise a profound influence on their conformations and functions. Since a direct measurement of the interfacial potential is rarely feasible, the electrophoretic mobility has become the most popular and widely accepted route to determining molecular charge.
Laser light scattering is prized for its ability to carry out non-destructive, first-principle measurements of macromolecules’ electrophoretic mobilities. However, when it comes to proteins, satisfactory results have been difficult to come by due to their small sizes (<5 nm) and their more conspicuous Brownian motions. Lengthier measurements are therefore necessary to average out the mobility-masking diffusion and reveal the macromolecular electrophoresis. In the process, these fragile molecules are subjected to electrical currents and often irreversibly damaged and degraded, rendering the results unreliable. As the solution ionicity increases, the situation deteriorates because even more current is required to drive measurable electrophoresis. Existing products on the market notoriously “cook” their protein samples and struggle to measure any macromolecule smaller than 5 nm at a reasonable concentration.
The key to the successful measurement of proteins’ mobilities lies in a much shortened measurement time and the availability of sufficient data to average away molecular diffusion. Wyatt Technology’s Möbiuζ achieves these goals through massive parallelism of detection and extends the measurable molecular size range below 1 nm. A reduced measurement time (<30 seconds in most cases) contributes to excellent preservation of precious and fragile protein samples.
In a specific example, Size Exclusion Chromatography (SEC) corroborated the preserved integrity of a sample of monoclonal antibody after the mobility measurement. A sample recovery rate of >98% was obtained. Another important advantage of Wyatt Technology’s Möbiuζ which results from the patent-pending detection process is its much increased detection sensitivity: 2 mg/mL lysozyme or 0.5 mg/mL BSA. This represents an order of magnitude greater sensitive than the closest competitor.
Simultaneous measurement of the macromolecular hydrodynamic radius is available with the WyattQELS™ option, which utilizes backward scattered light to determine the sample translational diffusion coefficient. Both reusable flow-through cells and disposable cells are offered for mobility (and QELS) measurements. (Stop-flow is required during mobility measurements.) Samples can be introduced by manual injection, an auto-sampler, syringe pump or an auto-titrator. The Möbiuζ also has temperature control capability and is able to perform automated temperature studies. Wyatt Technology’s patent-pending Atlas system pressurizes the Möbiuζ cell to 35 bar (500psi) for mobility measurements. This not only forces the evolved gas into the solution but also greatly reduces the volume of existing gas bubbles. Charge characterization of your samples in high salt buffers is no longer a daunting task.