Characterization of IgG Monomers & Their Aggregates
Application Note Feb 27, 2015
Malvern Instruments Limited
Whether we are trying to improve candidate formulations or trying to characterize final formulations, knowing exactly what a protein sample comprises and how much we have of each component is of paramount importance. Traditionally, the determination of such information has employed Size Exclusion Chromatography (SEC), otherwise known as Gel Permeation Chromatography (GPC). Using SEC, sample components can be identified by their molecular weights in a process that compares the migration of analyte molecules through a size exclusion or gel permeation column against that of a series of standards with known molecular weights. In addition, the relative amounts of each component can be simply extracted from the respective peak areas providing that a concentration detector is used to record the elution and the relevant extinction coefficients are known.
Although a well-established technique and an industry standard for the characterization of proteins, the use of column calibration SEC for the determination of molecular weight is not without issues. Principally amongst these are the inaccuracies introduced into the analyses through shape-dependent elution. In this application note we characterize two antibody samples using column calibration and multi-detection SEC. Here we show the outcomes of both analyses and demonstrate how multi-detection SEC - a technique that combines the resolving power of chromatography with the revealing power of light scattering detectors and a viscometer - provides more accurate data and a more complete characterization of the protein mixtures under study.