Top-Down and Middle-Down Mass Spectrometry of Antibodies

Abstract

Therapeutic antibodies, primarily immunoglobulin G-based monoclonal antibodies, are developed to treat cancer, autoimmune disorders, and infectious diseases. Their large size, structural complexity, and heterogeneity pose significant analytical challenges, requiring advanced characterization techniques. This review traces the 30-year evolution of top-down (TD) and middle-down (MD) mass spectrometry (MS) for antibody analysis, beginning with their initial applications and highlighting key advances and challenges throughout this period. TD MS allows for the analysis of intact antibodies, and MD MS performs analysis of the antibody subunits, even in complex biological samples. Both approaches preserve critical quality attributes such as sequence integrity, post-translational modifications (PTMs), disulfide bonds, and glycosylation patterns. Key milestones in TD and MD MS of antibodies include the use of structure-specific enzymes for subunit generation, the implementation of high-resolution mass spectrometers, and the adoption of non-ergodic ion activation methods such as electron transfer dissociation (ETD), electron capture dissociation (ECD), ultraviolet photodissociation (UVPD), and matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD). The combination of complementary dissociation methods and consecutive ion activation approaches has further enhanced TD/MD MS performance. The current TD MS record of antibody sequencing with terminal product ions is about 60% sequence coverage obtained using the activated ion-ETD approach on a high-resolution MS platform. Current MD MS analyses with about 95% sequence coverage were achieved using combinations of ion activation and dissociation techniques. The review explores TD and MD MS analysis of novel mAb modalities, including antibody-drug conjugates, bispecific antibodies, endogenous antibodies from biofluids, and immunoglobulin A and M-type classes.

Keywords: ADC; ETD; Orbitrap; antibody-drug conjugate; electron transfer dissociation; mAb; middle-down mass spectrometry; monoclonal antibody; proteoform; top-down mass spectrometry.

Graphical abstract

Fig. 1

Schematic overview of the different immunoglobulin (Ig) structures that have been studied with TD/MD MS: IgG1-4, IgA1, and IgM monomer. These Ig structures differ in disulfide bond connectivity and glycosylation sites and type. Red marks represent disulfide bonds and yellow marks indicate N-glycosylation sites. The IgG2-4 subclasses differ in the sequence of the heavy chain’s hinge region domains.

Fig. 2

Monoclonal antibody structural organization shown on the example of a NIST mAb. The structural components of the NIST mAb are depicted, showing the light chain (Lc), the heavy chain (Hc), the N-terminal part of the heavy chain (Fd), and the C-terminal part of the heavy chain (Fc/2) subunits, along with the Fab region and highlighted CDRs (137). Intra- and inter-chain disulfide bonds are marked in red. The expanded views outline the following structural features: (i) CDR3 of the heavy chain, (ii) the hinge region with cleavage sites for the IdeS and KGP enzymes, (iii) the N-terminus of the heavy chain modified with a pyroglutamate, (iv) the C-terminus of the heavy chain that is prone to the loss of Lys residue, and (v) the NST glycosylation sites on asparagine residues in the Fc region (yellow sticks) with complex N-linked glycans.

Fig. 3

Advances in mAbs sequence coverage annotation through diverse TD/MD MS approaches developed over the past 30 years. Each data point corresponds to an approach with a specific MS/MS technique(s), plotted by increasing sequence coverage on the x-axis. Each point corresponds to a reference entry in Tables 1 and 2 and is labeled by the MS method and associated table row number (e.g., #1.1 – row one in Table 1).

Fig. 4

Sequence coverage that was achieved for antibodies using TD-MS, MD-MS, and combinations of these approaches with various fragmentation techniques as a function of precursor ion molecular weight. These molecular weights correspond to common antibody subunits generated via structure-specific enzymes or disulfide bond reduction. F(ab′)₂ represents the ∼100 kDa fragment produced by IdeS digestion, containing two Lc, two Fd, and part of the hinge region. Each data point represents an experiment with a specific technique, positioned according to its resulting sequence coverage. Each point marks a specific experiment and is annotated with the method and table row number (e.g., #1.1 = Table 1, row 1).

Fig. 5

Sequence coverage (%) obtained for mAbs over the past 30 years using TD MS, MD MS, and combinations of these MS approaches with different fragmentation techniques. Each colored block represents a specific study, annotated with the applied MS method and the corresponding entry number from the review tables (e.g., #1.1 – row one in Table 1). The percentage of sequence coverage obtained in each study is displayed within its respective block.

Fig. 6

Research network graph generated based on co-authorship in TD and MD MS antibody analysis peer-reviewed publications. Each node symbolizes an individual researcher, with connecting lines indicating co-authorship links; stronger connections correspond to more frequent collaborations. A total of 62 research papers (listed in Table 1, Table 2, Table 3) involving 319 authors were analyzed, with 80 authors contributing to at least two papers on TD and MD MS antibody analysis. Distinct colors denote clusters of closely collaborating researchers. This network was generated using VOSviewer version 1.6.20 (138).