Mass Spectrometry Solutions to Characterizing Monoclonal Antibodies
Preparing mAb samples for MS analysis varies significantly depending upon whether the sample is enzymatically digested prior to use (bottom up and middle up) or examined intact (top down). In all cases it is usual to start with a purified sample. For intact (top-down) analyses, sample cleanup usually in the form of desalting is all that is required prior to analysis, hence reducing sample preparation and analysis times (see Section 2.3 for details on native (intact) sample preparation and conditions). Reducing agents such as dithiothreitol (DTT) and cleavage-specific enzymes such as immunoglobulin-degrading enzyme (from Streptococcus pyogenes) (IdeS), endoglycosidases, and PNGase F can be used to generate antibody subunit fragments between 25 and 50 kDa in size, with and without glycosylation (see Section 3.1 for more details). Middle-up approaches and MS analysis of smaller antibody fragments typically offer improved data quality and mass accuracy over intact analyses without significantly increasing the data analysis time. Liquid chromatography-tandem MS (LC-MS/MS) is frequently used for peptide mapping of mAbs to obtain structural characterization including glycan profiles for each individual glycosylation site .
A specialized form of bottom-up analysis is employed in HDX-MS utilizing an online pepsin column to digest the intact molecule, analyzing the resulting peptides to yield site-specific information. Examples of each type of analysis are detailed throughout the rest of this chapter.