Improved Characterization of IgG2 isoforms by LC-MS
2nd May 2019
By: Dr Martin De Cecco - Senior Scientist,
The majority of therapeutic mAbs developed so far belong to the IgG1 subclass, although some are IgG2 or IgG4. These different IgG subclasses differ in terms of the connectivity of their disulfide bonds. Whereas IgG1 molecules generally only have one defined structure, IgG2 antibodies can exist as three isoforms1, each with a different pattern of disulfide bonds (Figure 1).
The nature of the disulfide bonding in the IgG2 subclass has been shown to influence antigen binding activity in vitro.2 The heterogeneity associated with different disulfide isoforms adds to the structural complexity of IgG2, posing an additional challenge for the characterization of these molecules.
Reversed-phase liquid chromatography (LC) coupled to high-resolution mass spectrometry (MS) is a powerful analytical approach for the profiling of mAb therapeutics. The combination of high performance separations and accurate mass information can provide detailed insights into antibody heterogeneity and stability.3 However, to date, the highest resolution chromatographic separations of intact proteins have relied upon ion pairing reagents, which can compromise MS sensitivity.
To facilitate the characterization of IgG2 antibodies, we have developed a method that combines high resolution intact protein separations with high MS sensitivity. By exploiting recent advances in column technologies, we achieved excellent resolution of the different IgG2 isoforms. In fact, a number of additional species were detected. In addition, the use of a novel ion pairing reagent improved the MS sensitivity and enabled high quality mass spectra to be obtained for each species.
To find out more about the development of this method, download our scientific poster.
Figure 1. Schematic representation of the three isoforms of an IgG2 antibody showing the differences in inter-chain disulfide bond arrangements. Intra-chain disulfide bonds are not shown for clarity.
- Wypych et al., J. Biol. Chem. (2008) 283, 16194.
- M. Dillon et al., J. Biol. Chem. (2008) 283, 16206.
- M. Dillon et al. J. Chromatogr. A (2006), 1120, 112.