Wyatt Technology Develops New Automated Online Second Virial Coefficient (A₂) Method

Wyatt Technology Corporation, the world leader in absolute macromolecular characterization instrumentation and software announces that it has developed a pioneering new Static Light Scattering (SLS) method for measuring the Second Virial Coefficient (A₂), using its DAWN and miniDAWN light scattering and Optilab rEX refractometer instruments. A new application note details how the new Trainoff-Wyatt Online method simplifies and automates the A₂ measurement to help biochemists deliver high quality results in a fraction of the time and using much less single protein sample.

Protein-protein interactions are at the heart of virtually every process in a living cell. The more information that can be obtained about these interactions, the more that can be understood about diseases and ways can be found to treat them. Several methods exist for measuring protein interactions, including SLS and interaction chromatography. SLS is the preferred method since it is non-invasive and does not require modifying the sample in any way. The samples do not need to be tagged or immobilized on a surface.

The Second Virial coefficient (A₂) is a thermodynamic parameter used to characterize equilibrium solutions of weak non-specific protein-solvent interactions and is essential to understand a wide array of processes, such as stabilization of therapeutic protein formulations, purification of protein mixtures and crystallization of proteins. Both self and cross-association can be quantified by Virial coefficients. A₂ values are dependent on the protein and its solvent as well as temperature, salinity, pH and presence of chemical excipients. Results showing positive A₂ point to repulsive intermolecular interaction, while negative A₂ demonstrates attractive intermolecular interaction. The new method allows for rapid determination of buffer conditions required to tune A2 to a particular value. For example to develop a formulation buffer, a positive A₂ is required, whereas for crystallization, a small negative value is desirable. With the new method, many conditions can be tried and characterized until an optimal buffer composition is found.

Earlier methods of measuring A₂ required one to create multiple dilutions and to accurately dialyze the samples. This resulted in long experiment times and difficulty in producing reproducible results. The new method however, can produce results in a few minutes and uses 10% of the sample required by the traditional method. Moreover, it automatically dialyzes the sample eliminating much of the manual sample preparation.

For this particular application of the innovative Trainoff-Wyatt Online A₂ method, scientists used a standard autosampler to deliver a sequence of 5 or more different injection volumes that pass through an optional size exclusion chromatography column or a desalting dialysis column. This resulting eluant was detected by a Wyatt DAWN light scattering instrument and an Optilab rEX refractive index detector. The Optilab rEX served as a concentration detector and recorded the complete dialysis of each injection. This experiment can also be successfully carried out using a Wyatt mini-DAWN light scattering instrument.

The new method delivers accurate results at a greatly reduced cost. The data showed excellent agreement with the fit in part due to the automation of the process. Furthermore a single protein stock solution was used to produce results for an entire series of measurements. The method delivered the added benefit of flushing the light scattering flow cell between each injection, which is very helpful for researchers working with 'sticky' proteins.

For more information on Wyatt Technology’s new A₂ method or to obtain a copy of this application note, please visit the Article Webpage or contact Wyatt Technology by email.