Unique enzymes improve mass spectroscopy of IgG and make it possible to use affinity purification of acid sensitive IgG molecules (144.31)

Abstract The human pathogen Streptococcus pyogenes produces various surface proteins in order to avoid the host immune defence systems. Two of these proteins, EndoS and IdeS, have proven to be very useful as tools in antibody engineering. EndoS hydrolyses the chitobiose core of the aspargine linked glycan on the heavy chain of IgG and IdeS is a cysteine protease that cleaves the IgG molecule at a unique site in the hinge region. Here we show, for the first time, that mass spectrometric analysis of antibody fragments are greatly facilitated by combining EndoS and IdeS activities. Utilization of RPC18 LC-ESI-MS generated a well-defined protein envelope with a deconvoluted mass of 24137 Da corresponding to the IgG1 heavy chain cleaved at position Gly236. This opens up new possibilities for utilizing a combination of these enzymes for fast and accurate analysis of IgG:s in a variety of mass spectrometry applications. In addition, a mutated variant of the EndoS enzyme was found to show a high affinity for IgG molecules and has been evaluated in affinity purification of intact, native IgG molecules since it excludes non-native IgG molecules. Furthermore, the affinity purification using the mutant EndoS can be performed at neutral pH which is favourable in purification of acid sensitive IgG clones. The performance and characteristics of Ides, EndoS and the EndoS mutant is highlighted during the presentation with data from several individual studies.