Footprinting for fingerprinting: proof-of-concept for the use of hydroxyl radical protein footprinting for structural comparison studies

Abstract

Monoclonal antibodies (mAbs) require extensive physicochemical characterization to ensure product quality. The clinical and pharmacological properties of mAbs are linked to higher order structure (HOS), but high-resolution analytical techniques to evaluate structural conformations and structural comparisons during drug design and process development are limited. Here, we provide proof-of-concept for the use of hydroxyl radical footprinting-mass spectrometry (HRPF-MS) to characterize the average solvent accessible surface area ( < SASA>) of mAbs. The premise is that each mAb exhibits a unique "oxidative footprint" that may aid in demonstrating structural similarities and differences between mAbs and support the HOS characterization of a biotherapeutic fingerprint. This work, which includes case studies for comparing oxidative footprints between mAbs, highlights the challenges and future state needed to realize the potential of HRPF for the application of biotherapeutic fingerprinting.

Keywords: Hydroxyl radical protein footprinting – mass spectrometry; biotherapeutic fingerprint; monoclonal antibody; oxidative footprinting; structural consistency.

Figure 1.

Comparison of the oxidative footprints from five IgG1 molecules (mAb1–5) and one IgG4 molecule (mAb6).

Figure 2.

Comparison of the oxidative footprint. (a) Fc-region comparison of five IgG1 molecules. (b) Fc-region comparison of two IgG molecules with similar glycan heterogeneity. (c) oxidative footprint comparison of the native form vs. afucosylated form of an IgG1 molecule. (d) oxidative footprint of the native form vs. YTE-mutation form of an IgG4 molecule. *denotes peptides with likely practically significant differences (dark blue vs. light blue bars).

Figure 3.

Comparison of the oxidative footprint of mA1 reference batch, process 1 batches, and process 2 batches. *denotes hotspot peptides with propensity for degradation.

Figure 4.

Structure–function study of impact on light stress to mAb5. (a) Light stress impacts ADCC but not the associated antigen and receptor binding. (b) Comparison of control vs. light stress oxidative footprinting reveals structural differences both proximal and distal to the FcγRIIIa binding site. (c) Visual representation of structural impact based on HRPF data. *denotes peptides with likely practically significant differences. N.A denotes peptides heavily oxidized from stress exposure and not appropriate for comparison.