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Measuring the 2nd Virial Coeffient and Molecular Density of Proteins to Improve Crystallization

Measuring the 2nd Virial Coeffient and Molecular Density of Proteins to Improve Crystallization content piece image
One of the challenges in biopharmaceutical development is measuring the 3-d structure of the active protein. The diffraction of X-ray beams through a crystalline structure can be measured to produce a 3-d picture of the density of electrons within a sample. From this the position of atoms in that sample as well as their chemical bonds can be determined. X-ray crystallography is able to help identify the structure and therefore the function of the active protein. A key issue with this technique is the necessity for the sample to be in a crystalline form. With proteins the crystallisation process can be difficult, as solvent conditions have a significant impact on the ability of a sample to form crystals.

The second virial coefficient (B22 or A2) is a thermodynamic parameter which describes the interaction strength between a protein and a solvent, and can be correlated with protein solubility. This parameter is used as an indicator of the potential for a protein to crystallise within a given solvent. Negative virial coefficient values are indicative of attractive interactions, with macromolecules tending to self-associate. Positive virial coefficient values are indicative of repulsive interactions, with the macromolecules preferring solvation.

It is possible to use Static Light Scattering (SLS) detectors to measure the second virial coefficient of a protein. This parameter can be measured either using a batch SLS detector or by a Size Exclusion Chromatography (SEC) linked SLS detector.

In this poster a Malvern Instruments SEC-MALS 20 detector was connected to a Refractive Index (RI) concentration detector and an online differential viscometer (DP). This has the advantage of not only measuring the protein molecular weight, and subsequently the second virial coefficient, but also the Intrinsic Viscosity (IV) of a sample. The IV is inversely proportional to the molecular density of a sample so can be used to provide information about the samples conformation as well as allowing the hydrodynamic radius (Rh) to be calculated.

The second virial coefficient of Human Serum Albumin (HSA) has been measured using two different buffer conditions, Phosphate buffer pH 7.4 and Citrate buffer pH 4.1. Interesting and contrasting data was obtained for these two buffer conditions