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 proxy for molecular charge.
As well as being a non-invasive method, laser light scattering is prized for its ability to carry out physical, 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 5nm 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.
New mobility instruments achieve these goals through massive parallelism of detection and extend the measurable molecular size range below 2nm (Fig. 1). A reduced measurement time (<60 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 per cent was obtained using Wyatt Technology's Möbiu nobility instrument (Fig. 2). Another important advantage of recently developed mobility instruments which results from the patent-pending detection process is its much increased detection sensitivity: 2mg/mL lysozyme, or 0.5mg/mL BSA. This represents an order of magnitude greater sensitivity than the closest competitor.
With the new instrumentation, simultaneous measurement of the macromolecular hydrodynamic radius is available when combined with quasi-elastic light scattering, which utilises backward scattered light to determine the sample translational diffusion coefficient.
Both reusable flow-through cells and disposable cells can be employed 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 Möbiu also has temperature control capability and is able to perform automated temperature studies.
- Vincent Hsieh is R&D Scientist with Wyatt Technology, Santa Barbara, California, USA. www.wyatt.com.
