Characterization of Proteins and Protein Self Association (Oligomerization) with SEC-MALS
Poster Oct 08, 2014
Michel Terray, Mark Pothecary
As the biopharmaceutical market continues to grow at a faster rate than the small molecule pharmaceutical market, the requirement for tools for the detailed characterization of proteins is also growing. Of particular interest is the requirement to characterize protein self-association, or oligomerization. Size exclusion chromatography is routinely used as a tool for monitoring oligomerization but can be inexact when using a single detector. Single detector chromatography can work well for globular proteins that conform to a typical column calibration molecular weight curve. However, oligomers (and indeed some monomers) are not necessarily globular in structure and thus their molecular weight cannot be accurately determined using this method.
Since light scattering detectors were introduced, they have seen several different development iterations. Modern systems are now either based on the RALS/LALS single angle approach or the MALS (multi-angle) approach. All are capable of measuring absolute molecular weight of proteins, independent of their retention volume. This allows oligomers and larger aggregates to be easily identified based on their light scattering signal which relates directly to their molecular weight. A light scattering detector must be combined with a concentration detector. While UV is the most commonly used detector for measuring protein concentration, a refractive index detector has advantages. In particular, protein dn/dc (proteins refractive index increment) is approximately constant, especially when compared to dA/dc (proteins UV response) which is highly variable. This significantly simplifies the concentration and molecular weight measurement.
In this poster, a series of proteins and their oligomers were characterized using SEC-MALS with UV and RI detection. The monomer molecular weights are measured and compared with those from column calibration. The molecular weights of their oligomers are also compared with column calibration and the differences explored. It can be seen that the use of light scattering detectors, significantly improves the accuracy of the molecular weight and thus the confidence in identifying populations as particular oligomers.