Inter-laboratory Study of mAb Characterisation using Top-Down and Middle-Down Mass Spectrometry

September 15, 2020 | Applications, References |

Srzentic et al. present an interlaboratory study investigating the current state of top-down and middle-down mass spectrometry methods for the characterization of therapeutic monoclonal antibodies and explore the potential added value of these methods to the more commonly used bottom-up mass spectrometry.


In the growing market of therapeutic monoclonal antibodies (mAbs), there is an increasing demand to precisely and reproducibly characterize the structural heterogeneity of mAbs. At present, bottom-up mass spectrometry (MS) approaches, using peptide mapping of tryptic peptides, is widely employed for this. In this study, initiated by the Consortium of Top-Down Proteomics, the use of top-down and middle-down MS methods were explored as potential complements to bottom-up MS approaches in regard to primary structure confirmation, variable domain sequencing and post-translational modification (PTM) identification on mAbs.


The three commercial mAbs: SiLuLite, NIST mAb standard and Herceptin were centrally prepared in three different forms before being distributed to 20 participating laboratories worldwide; intact for top-down analysis, and digested using either of the IgG proteases GingisKHAN (above the hinge) or FabRICATOR (below the hinge) for middle-level analysis. The participating laboratories were asked to analyze these using their top-down/middle-down MS workflow of choice.


The study revealed the wide variety of techniques and expertise available among the participants. Nevertheless, top-down/middle-down approaches proved to be a fast and valuable compliment to bottom-up approaches in achieving a high amino acid sequence coverage, avoiding artifacts and confirming bottom-up derived PTM profiling and stoichiometry. They could also deduce if protein modifications observed in bottom-up assays were naturally occurring or sample preparation mediated. Middle-level analysis, using FabRICATOR or GingisKHAN generated subunits, proved especially beneficial for assigning correct monoisotopic masses and identifying glycosylation and glycation sites. The use of IgGZERO to remove the Fc N-glycans and eliminate glycosylation related heterogeneity prior to top-down/middle-down MS analysis was also suggested. Overall, the study shows the added value of top-down/middle-down approaches for mAb analysis and the great potential for further advances in top-down and middle-down MS techniques.


Link to Paper: Srzentić et al., 2020. Inter-laboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry. Anal. Chem. doi: 10.1021/jasms.0c00036