Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies

Abstract

Monoclonal antibodies (mAbs) are powerful therapeutics, and their characterization has drawn considerable attention and urgency. Unlike small-molecule drugs (150-600 Da) that have rigid structures, mAbs (∼150 kDa) are engineered proteins that undergo complicated folding and can exist in a number of low-energy structures, posing a challenge for traditional methods in structural biology. Mass spectrometry (MS)-based biophysical characterization approaches can provide structural information, bringing high sensitivity, fast turnaround, and small sample consumption. This review outlines various MS-based strategies for protein biophysical characterization and then reviews how these strategies provide structural information of mAbs at the protein level (intact or top-down approaches), peptide, and residue level (bottom-up approaches), affording information on higher order structure, aggregation, and the nature of antibody complexes.

Keywords: FPOP; Hydrogen/deuterium exchange; Ion mobility; Mass spectrometry; Monoclonal antibody; Native ESI; Protein footprinting; Top-down and bottom-up.

Figure 1

IgG structure (IgG1). The global structure of IgG1 has two identical heavy chains and light chains. Four chains are attached covalently with inter-chain disulfide bonds and also by non-covalent interactions. The two constant regions from heavy chains (CH2 and CH3, Fc regions) respond to the binding to Fc gamma and FcRn receptors. The variable region from both light and heavy chains contains antigen-binding regions (CDRs). Variable regions with the close constant region together are called the Fab region. The Fab and Fc region are linked by the hinge region in the heavy chain. In the IgG1, there is a glycosylation site on the second constant region (CH2).

Figure 2

Overview of top-down and bottom-up MS based protein biophysical studies. The left circle is the summary of top-down approaches. The right circle is the summary of bottom-up approaches.

Figure 3

Native ESI, IM and ECD mass spectra of WT IgG2, (a) ion mobility separation, (b) ECD top-down with in-source activation, and (c) highlighted region in yellow of light chain in CDR showing ECD cleavage sites. (Copied with permission from JASMS)

Figure 4

The MS based biophysical “tool box” in characterizations of IgG2 with different S-S bond networks. This is an example of combining multiple approaches, not limited to MS, to provide complementary structural information of mAbs. The experimental time scale and structural resolution are labeled.