Rapid LC-MS screening for IgG Fc modifications and allelic variants in blood

A new method for simultaneously screening allelic variants and certain Fc modifications on endogenous human IgG1 and IgG2 directly from blood samples is described. The IdeS endoproteinase was used to cleave IgG in serum to generate Fc, which, after denaturation, was analyzed directly as monomeric Fc (Fc/2) by LC-MS to identify the haplotype(s) present in each individual. The relative levels of IgG isotype and haplotype ratios were generated along with the profile of the major Fc glycans and several other modifications associated with each IgG1 or IgG2 haplotype. Since only minute quantities (5 μL) of blood are required and analysis can be highly automated, this approach lends itself to screening large populations. We demonstrate its utility in examining possible correlations between Fc properties and allelic variants. IgG1 core fucosylation, which significantly impacts antibody dependent cellular cytotoxicity (ADCC), showed an asymmetric distribution, with a small number of individuals showing unexpectedly high core afucosylation levels. In these individuals, IgG2 afucosylation levels were normal. Finally, a new IgG1 allotype, previously not characterized, was identified using this analytical methodology.