Comprehensive glycosylation profiling of IgG and IgG-fusion proteins by top-down MS with multiple fragmentation techniques

Abstract

We employed top-and middle-down analyses with multiple fragmentation techniques including electron transfer dissociation (ETD), electron capture dissociation (ECD), and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) for characterization of a reference monoclonal antibody (mAb) IgG1 and a fusion IgG protein. Fourier transform ion cyclotron resonance (FT-ICR) or high performance liquid chromatography electrospray ionization (HPLC-ESI) on an Orbitrap was employed. These experiments provided a comprehensive view on the protein species; especially for different glycosylation level in these two proteins, which showed good agreement with oligosaccharide profiling. Top-and middle-down MS provided additional information regarding glycosylation sites and different combinational protein species that were not available from oligosaccharide mapping or conventional bottom-up analysis. Finally, incorporating a limited enzymatic digestion by immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS) with MALDI-ISD analysis enabled extended sequence coverage of the internal region of protein without pre-fractionation. Biological significance: Oligosaccharide profiling together with top-and middle-down methods enabled: 1) detection of heterogeneous glycosylated protein species and sites in intact IgG1 and fusion proteins with high mass accuracy, 2) estimation of relative abundance levels of protein species in the sample, 3) confirmation of the protein termini structural information, and 4) improved sequence coverage by MALDI-ISD analysis for the internal regions of the proteins without sample pre-fractionation.

Ion Activation Methods for Peptides and Proteins

“…219 In another study, the fragment ion information from both ECD and ETD activation methods and from both top-down and middle-down approaches was combined to analyze several IgG fusion proteins, ultimately obtaining sequence coverage of 61%. 220 …”

Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer

“…It also allows the identification and localization of major modifications and the characterization of major glycoforms. [16] It has the advantage of requiring a highly simplified sample preparation and therefore minimal artifact introduction induced by sample processing. Despite these advantages, TD MS is not yet routinely used for the characterization of mAbs, primarily due to the large number of highly charged products yielded by IgG fragmentation, which requires high-resolution MS and the applications of techniques aimed at improving sensitivity, such as spectral summation (averaging).…”

Structural characterization of expressed monoclonal antibodies by single sample mass spectral analysis after IdeS proteolysis

“…Several somewhat similar methods for analysis of monoclonal antibodies utilizing IdeS protease and mass spectral analyses have been published recently (An et al [4], Tran et al [5] Reusch et al [6] Janin-Bussat et al [7] Chevreux et al [8]). However, unlike these previously published procedures, we alkylated the reduced monoclonal antibody (with iodoacetamide) prior to mass spectral analyses, in order to eliminate the possibility of any further cysteine modifications or oxidation leading to disulfide formation prior to analysis.…”