A simple MS method to characterize the higher order structures of antibody therapeutics

A scheme with enhanced throughput using the hydroxyl radical foot-printing technique to map the higher order structures (HOSs) of antibody therapeutics was developed. This method takes advantage of the simplicity of multiple reaction monitoring (MRM) by monitoring the remaining unmodified peptides, thus overcoming the complexity of the data analysis of the conventional free radical foot-printing technique, wherein various oxidized forms of the peptides are measured. In this study, a preliminary method validation was carried out to show that the technique has good specificity, sensitivity, and repeatability. Finally, we were able to use the method to differentiate the HOSs of the disulfide isoforms of the IgG2 antibody, provide site-specific structural differences between IgG1 and its deglycosylated counterpart, and determine similarities between a biosimilar candidate and the originator product.