Direct-MS analysis of antibody-antigen complexes

Abstract

In recent decades, antibodies (Abs) have attracted the attention of academia and the biopharmaceutical industry due to their therapeutic properties and versatility in binding a vast spectrum of antigens. Different engineering strategies have been developed for optimizing Ab specificity, efficacy, affinity, stability and production, enabling systematic screening and analysis procedures for selecting lead candidates. This quality assessment is critical but usually demands time-consuming and labor-intensive purification procedures. Here, we harnessed the direct-mass spectrometry (direct-MS) approach, in which the analysis is carried out directly from the crude growth media, for the rapid, structural characterization of designed Abs. We demonstrate that properties such as stability, specificity and interactions with antigens can be defined, without the need for prior purification.

Keywords: antibody antigen interactions; antibody deign; native mass spectrometry; protein stability.

Figure 1 –. CIU plots reflect the differences in gas-phase stability between the parental and designed G6 Abs.

The two Abs were reduced with TCEP and subjected to CIU analysis. Drift time values were extracted from the apex of each MS peak and plotted as a function of collision voltage. Data is shown for the 21⁺, 22⁺ and 23⁺ charge states. The overall unfolding pattern of the two Abs is similar and consists of two transition states. However, the increase in acceleration voltage leads to larger shifts in drift time values of the parental Ab in comparison the designed G6^des13, indicating increased gas-phase stability of the latter.

Figure 2 –. Ab/antigen interactions can be probed in the context of the crude growth medium, as demonstrated for the anti-lysozyme D44.1^des Ab.

(A) Increasing concentrations of lysozyme were titrated into the D44.1^des growth medium, giving rise to the gradual occupation of the Ab’s binding sites. An extended view of the 6360–6540 m/z range is shown. The additional charge state series seen to the right of the D44.1^des peaks, correspond to a glycoform with a 620 Da mass shift, indicating the addition of two fucose and two mannose moieties. (B) Quantification of the relative abundance of the unbound (blue line), single-bound (orange line) and double-bound D44.1^des (grey line) Abs. The results are averaged over three independent measurements. Error bars represent standard deviations.

Figure 3 –. Revealing the Ab/antigen buried surface area of interaction in crude media.

IM-MS plots (left panel) and the extracted m/z spectra (right panel) of (A) D44.1^des, (B) lysozyme and (C) the saturated D44.1^des/lysozyme₂ complex. (D) High-resolution structure of the D44.1 Fab variant bound to lysozyme (left panel) and CCS values of free D44.1^des, lysozyme and their D44.1^des/lysozyme2 complex, as well as of buried surface area, as extracted from IM-MS measurements and PISA calculations using the crystal structure (1P2C) (right panel).

Figure 4 -. The monoclonal MCP21 Ab is specific to human 20S proteasome and does not bind the rat and yeast proteasomes.

(A) Mass spectrum of the human 20S proteasome mixed with the MCP21 hybridoma growth medium, wherein multiple proteasome-Ab species are detected. In contrast, the rat (B) or yeast (C) 20S proteasomes did not interact with the MCP21 Ab, validating the specificity of the Ab towards human complexes. All measured masses are shown in Supplementary Table 1.