Automated, On-line Second Virial Coefficient Measurements

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The Application Notebook

The Application Notebook, The Application Notebook-09-02-2007, Volume 0, Issue 0

Measurement of weak protein-solvent interactions is essential in 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 the second virial coefficient (A2).

A2 values are dependent on both the protein and its solvent: Temperature, salinity, pH, chemical excipients, and so on, can induce dramatic shifts in A2. Positive A2 corresponds to repulsive intermolecular interaction, whereas negative A2 corresponds to attractive intermolecular interaction. Rapid determination of the buffer conditions yielding an optimal A2, such as a positive A2 for formulations, or a slightly negative A2 for crystallization, is highly desirable.

Figure 1

In the past, such measurements were labour intensive, time consuming and expensive in terms of time and protein: For each buffer condition tested, extensive dialysis and preparation of multiple concentrations were required. Additionally, each concentration had to be injected manually. Wyatt Technology has overcome these issues, and developed an accurate, automated method for measuring A2 on-line. In our on-line A2 method, only a single protein stock solution is needed for an entire series of measurements.

In addition to complete automation and on-line dialysis, the on-line A2 method has the added benefit of flushing the light scattering flow cell between each injection, helpful for researchers working with "sticky" proteins.

The Trainoff-Wyatt On-line A2 method is as follows: A sample delivery unit, such as a standard autosampler, delivers a sequence of 5 or more different volumes (such as 20 μ to 200 μL) through an optional size exclusion chromatography column (SEC) or a desalting column, to a DAWN or miniDAWN light scattering instrument and an Optilab RI. The Optilab RI serves as a concentration detector and also records the complete dialysis of each injection.

Figure 2

The On-line A2 method will enable researchers to fully use the second viral coefficient as an important indicator of protein–protein interaction. The historic impediments to this type of measurement, lengthy sample preparation and experimental labour, have been overcome. Now, using standard chromatography equipment and Wyatt detectors, an entire series of A2 values can be determined in less then a day!

DAWN, miniDAWN, ASTRA, Optilab and the Wyatt Technology logo are registered trademarks of Wyatt Technology Corporation. ©2007 Wyatt Technology Corporation 7/20/07

Amy Hanlon, Wyatt Technology Corp., Santa Barbara, California, USA.

Wyatt Technology Corporation

6300 Hollister Avenue, Santa Barbara, California 93117, USA

tel. +1 805 681 9009 fax +1 805 681 0123

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